MEDICAL 


THE 

VITAMINE  MANUAL 

A  Presentation  of  Essential  Data 

About  the 
New  Food  Factors 


BY 

WALTER  H.  EDDY 

to 

ASSOCIATE  PROFESSOR  PHYSIOLOGICAL  CHEMISTRY 
Teachers  College,  Columbia  University 


BALTIMORE 

WILLIAMS  &  WILKINS  COMPANY 

1921 

'"' 


COPYRIGHT  1921 
WILLIAMS  &  WILKINS  COMPANY 

Made  in  United  States  of  America 

All  rights  reserved,  including  that  of  translation 

into  foreign  languages,  including 

the  Scandinavian. 


COMPOSED   AND  PRINTED   AT  THE 

WAVERLY  PRESS 

BY  THE  WILLIAMS  &  WILKINS  COMPANY 

BALTIMOBE,  MD.,  U.  S.  A. 


CONTENTS 

CHAPTER  I 
How  VITAMINES  WERE  DISCOVERED 7 

CHAPTER  II 
THE  ATTEMPTS  TO  DETERMINE  THE  CHEMICAL  NATURE  OP  A  VITAMINE.    25 

CHAPTER  III 
METHODS  USED  IN  TESTING  FOR  VITAMINES 32 

CHAPTER  IV 
THE  YEAST  TEST  FOR  VITAMINE  B 51 

CHAPTER  V 
THE  SOURCES  OP  THE  VITAMINES 56 

CHAPTER  VI 
THE  CHEMICAL  AND  PHYSIOLOGICAL  PROPERTIES  OP  THE  VITAMINES.  .    62 

CHAPTER  VII 
How  TO  UTILIZE  THE  VITAMINES  IN  DiEts 82 

CHAPTER  VIII 

AVITAMINOSES    OR     THE     DISEASES     THAT     RESULT    FROM     VlTAMINE 

DEFICIENCIES 89 

CHAPTER  IX 
BIBLIOGRAPHY..  94 


967  J 


PREFACE 

The  presentation  of  essential  data  concerning  vitamines  to  suc- 
ceeding groups  of  students  has  become  increasingly  difficult  with 
the  development  of  research  in  this  field.  The  literature  itself  has 
assumed  a  bulk  that  precludes  sending  the  student  to  original 
sources  except  in  those  instances  when  they  are  themselves  to 
become  investigators.  The  demand  on  the  part  of  the  layman  for 
concise  information  about  the  new  food  factors  is  increasing  and 
worthy  of  attention.  For  all  of  these  reasons  it  has  seemed  worth 
while  to  collate  the  existing  data  and  put  it  in  a  form  which  would 
be  available  for  both  student  and  layman.  Such  is  the  purpose 
of  this  little  book. 

It  has  been  called  a  manual  since  the  arrangement  aims  to  pro- 
vide the  student  with  working  material  and  suggestions  for  inves- 
tigation as  well  as  information.  The  bibliography,  the  data  in 
the  chapter  on  vitamine  testing,  the  tables  and  the  subdivision  of 
subject  matter  have  all  been  arranged  to  aid  the  laboratory  workers 
and  it  is  the  hope  that  this  plan  may  make  the  manual  of  especial 
value  to  the  student  investigator.  The  management  also  sepa- 
rates the  details  necessary  to  laboratory  investigation  from  the 
more  purely  historical  aspects  of  the  subject  which  we  believe  will 
be  appreciated  by  the  lay  reader  as  well  as  the  student. 

No  apologies  are  made  for  data  which  on  publication  shall  be 
found  obsolete.  The  whole  subject  is  in  too  active  a  state  of  inves- 
tigation to  permit  of  more  than  a  record  of  events  and  their  appar- 
ent bearing.  Whenever  there  is  controversy  the  aim  has  been  to 
cite  opposing  views  and  indicate  their  apparent  value  but  with  full 
realization  that  this  value  may  be  profoundly  altered  by  new  data. 

Since  the  type  of  the  present  manual  was  set,  Drummond  of 
England  has  suggested  that  we  drop  the  terminal  "e"  in  Vita- 
mine,  since  the  ending  "ine"  has  a  chemical  significance  which  is 
to  date  not  justified  as  a  termination  for  the  name  of  the  un- 
identified dietary  factors."  This  suggestion  has  been  generally 
adopted  by  research  workers  and  the  spelling  now  in  use  is 
Vitamin  A,  B,  or  C.  It  has  hardly  seemed  worth  while  to  derange 

5 


6  PREFACE 

the  entire  set  up  of  the  present  text  to  make  this  correction  and 
we  have  retained  the  form  in  use  at  the  time  the  mamuscript  was 
first  set  up.  The  suggestion  of  Drummond,  however,  is  sound 
and  will  undoubtedly  be  generally  adopted  by  the  research  work- 
ers in  the  subject. 

Attempt  has  been  made  to  cover  all  the  important  contributions 
up  to  April,  1921.  Opportunity  has  permitted  the  inclusion  of 
certain  data  of  still  later  date  and  undoubtedly  other  important 
papers  of  earlier  date  will  have  been  overlooked. 

It  is  a  pleasure  to  acknowledge  the  assistance  received  in  the 
preparation  of  the  manuscript  from  Dr.  H.  C.  Sherman,  Dr.  Mary 
S.  Rose  and  Dr.  Victor  La  Mer.  Their  suggestions  have  been 
most  valuable  and  greatly  appreciated. 

WALTER  H.  EDDY. 
Department  of  Physiological  Chemistry, 
Teachers  College,  Columbia  University, 
New  York  City,  April,  1921 


CHAPTER  I 

How  VITAMINES  WERE  DISCOVERED 

In  1911  Casimir  Funk  coined  the  name  Vitamine  to  describe 
the  substance  which  he  believed  curative  of  an  oriental  disease 
known  as  beri-beri.  This  disease  is  common  in  Japan,  the  Phil- 
ippines and  other  lands  where  the  diet  consists  mainly  of  rice, 
and  while  the  disease  itself  was  well  known  its  cause  and  cure 
had  baffled  the  medical  men  for  many  years.  Today  in  maga- 
zines, newspapers  and  street  car  advertisements  people  are  urged 
to  use  this  or  that  food  or  medicament  on  the  plea  of  its  vitamine 
content.  In  less  than  ten  years  the  study  of  vitamines  has 
increased  to  such  an  extent  that  it  is  difficult  to  find  a  chemical 
journal  of  any  month  of  issue  that  does  not  contain  one  or  more 
articles  bearing  on  the  subject.  Such  a  rapid  rise  to  public  notice 
suggests  an  importance  that  justifies  investigation  by  the  laity  as 
well  as  the  chemist  and  in  the  pages  that  follow  has  been  out- 
lined in  simple  language  the  biography  of  this  newest  and  lustiest 
of  the  chemist's  children. 

Dr.  Funk  christened  one  individual  but  the  family  has  grown 
since  1911  to  three  members  which  for  lack  of  better  names  are 
now  called  vitamines  "A,"  "B,"  and  "C."  There  are  now 
rumors  of  another  arrival  and  none  dare  predict  the  limits  of  the 
family.  Had  these  new  substances  been  limited  to  their  relation 
to  an  obscure  oriental  disease  they  would  have  of  course  com- 
manded the  medical  attention  but  it  is  doubtful  whether  the 
general  public  would  have  found  it  worth  while  to  concern  them- 
selves. It  is  because  on  better  acquaintance  they  have  com- 
pelled us  to  reform  our  ideas  on  nutrition  of  both  adults  and 
babies  and  pick  out  our  foods  from  a  new  angle,  that  we  accord 
them  the  attention  they  demand  and  deserve.  Granting  then, 
their  claim  upon  our  attention,  let  us  review  our  present  knowl- 
edge and  try  to  see  with  just  what  we  are  dealing.  This  will  be 
more  easily  accomplished  if  we  consider  the  vitamines  first  from 
the  historical  side  and  reserve  our  attention  to  details  of  behavior 

until  later. 

7 


8  VITAMINE   MANUAL 

A  limited  diet  of  polished  rice  and  fish  is  a  staple  among  the 
peoples  of  the  Orient.  When  the  United  States  Government 
took  over  the  Philippine  Islands  in  1898  it  sent  there  a  small 
group  of  scientists  to  establish  laboratories  and  become  acquainted 
with  the  peculiarities  of  the  people  and  their  troubles.  One  of 
the  first  matters  that  engaged  their  attention  was  the  condition  of 
the  prisons  which  were  most  unsanitary  and  whose  inhabitants 
were  poorly  fed  and  treated.  Reforms  were  put  into  operation 
at  once  and  the  sanitary  measures  soon  changed  these  prisons  to 
places  not  quite  so  abhorrent  to  the  eye.  In  trying  to  improve 
the  diets  of  the  prisoners  little  change  was  made  in  their  compo- 
sition because  of  the  native  habits  but  the  reformers  saw  to  it 
that  the  rice  fed  should  be  clean  and  white.  In  spite  of  these 
measures  the  first  year  saw  a  remarkable  increase  in  the  disease 
of  beri-beri,  and  the  little  group  of  laboratory  scientists  had  at 
once  before  them  the  problem  of  checking  a  development  that  bid 
fair  to  become  an  epidemic.  In  fact,  the  logical  discoverers  of 
what  we  now  know  as  the  antineuritic  vitamine  or  vitamine  "B" 
should  have  been  this  same  group  of  laboratory  workers  for  it 
was  largely  due  to  their  work  between  the  years  1900  and  1911 
that  the  ground  was  prepared  for  Funk's  harvest. 

The  relation  of  rice  to  this  disease  was  more  than  a  suspicion 
even  in  1898.  In  1897  a  Dutch  chemist,  Eijkman,  had  succeeded 
in  producing  in  fowls  a  similar  set  of  symptoms  by  feeding  them 
with  polished  rice  alone.  This  set  of  symptoms  he  called  poly- 
neuritis  and  this  term  is  now  commonly  used  to  signify  a  beri- 
beri in  experimental  animals.  Eijkman  found  that  two  or  three 
weeks  feeding  sufficed  to  produce  these  symptoms  and  it  was 
he  who  first  showed  that  the  addition  of  the  rice  polishings  to  the 
diet  was  sufficient  to  relieve  the  symptoms.  Eijkman  first 
thought  that  the  cortical  material  contained  something  necessary 
to  neutralize  the  effects  of  a  diet  rich  in  starch.  Later  however, 
he  changed  his  view  and  in  1906  his  position  was  practically  the 
view  of  today.  In  that  same  year  (1906)  F.  Gowland  Hopkins 
in  England  had  come  to  the  conclusion  that  the  growth  of  labora- 
tory animals  demanded  something  in  foods  that  could  not  be 
accounted  for  among  the  ordinary  nutrients.  He  gave  to  these 
hypothetical  substances  the  name  "accessory  food  factors." 


HOW  VITAMINES   WERE   DISCOVERED  9 

To  Hopkins  and  to  Eijkman  may  therefore  be  justly  attributed 
the  credit  of  calling  the  world's  attention  to  the  unknown  sub- 
stances which  Funk  was  to  christen  a  little  later  with  the  name 
vitamines.     Other  workers,  of  course,  knew  of  these  experiments 
of  Eijkman  and  Hopkins  and  in  1907  two  of  them,  Fraser  ano^ 
Stanton,  reported  that  by  extracting  rice  polishings  with  alcohol 
they  had  secured  a  product  which  if  added  to  the  diet  of  a  sufferer 
from  beri-beri  seemed  to  produce  curative  effects.     It  is  obvious 
that  logic  would  have  decreed  that  some  of  these  workers  should 
be  the  ones  to  identify  and  name  the  curative  material.     But 
history  is  not  bound  by  the  rules  of  logic  and  it  was  so  in  this 
case.     Another  student  had  been  attracted  to  the  problem  and 
was  working  at  the  time  in  Germany  where  he  also  became 
acquainted  with  Eijkman's  results  and  began  the  investigation 
of   rice    polishings    on    experimental   lines.     This   student    was 
Casimir  Funk  and  a  little  later  he  carried  his  studies  to  England 
where  he  developed  the  results  that  made  him  the  first  to  announce 
the  discovery  of  the  unknown  factor  which  he  christened  vita- 
mine.    Funk's  studies  combined  a  careful  chemical  fractioning 
of  the  extracts  of  rice  polishings  with  tests  for  their  antineuritic 
power  upon   polyneuritic   birds,   after  the  manner  taught   by 
Eijkman.     By    carrying    out    this   fractioning    and    testing    he 
obtained  from  a  large  volume  of  rice  polishings  a  very  small 
amount  of  a  crystalline  substance  which  proved  to  be  curative  to 
a  high  degree.     A  little  later  he  demonstrated  that  this  same 
substance  was  particularly  abundant  in  brewers'  yeast.      From 
these  two  sources  he  obtained  new  extracts  and  carefully  repeated 
his  analytical  fractionings.     The  result  was  the  demonstration 
that  they  contained  a  substance  which   could  be  reduced  to 
crystalline  form  and  was  therefore  worthy  of  being  considered  a 
chemical  substance.     In  1911,  before  Fraser  and  Stanton  or  any 
other  workers  had  been  able  to  show  to  what  their  curative 
extracts  were  due,  Funk  produced  his  product,  demonstrated  its 
properties  and  claimed  his  right  to  naming  the  same.     At  that 
he  barely  escaped  priority  from  still  another  source.     The  chem- 
ists in  Japan  were  naturally  interested  in  this  problem  and  pos- 
sessed an  able  worker  by  the  name  of  Suzuki.     Suzuki  and  his 
co-workers  Odake  and  Shimamura  were  engaged  in  the  same 


10  VITAMINE  MANUAL 

fractioning  processes  with  polishings  and  entirely  independently 
of  Funk  or  other  workers  they  too  succeeded  in  isolating  a  cura- 
tive substance  and  published  their  discovery  the  same  year  as 
Funk,  1911.  Their  methods  v/ere  later  shown  to  be  identical 
up  to  a  certain  point.  Suzuki  called  his  product  "Oryzanin." 
Funk's  elementary  analyses  had  shown  the  presence  of  nitrogen 
in  this  product  and  his  method  of  extraction  indicated  that  this 
nitrogen  was  present  in  basic  form.  For  that  reason  he  sug- 
gested that  his  product  belonged  to  a  class  of  substances  which 
chemists  call  "amines."  Since  its  absence  meant  death  and  its 
presence  life  what  more  natural  than  to  call  it  the  Life-amine  or 
Vita-amine.  This  is  the  origin  of  Funk's  nomenclature. 

Both  Funk's  original  crystals  and  Suzuki's  oryzanin  were  later 
shown  to  be  complexes  of  the  curative  substances  combined  with 
adulterants  and  we  do  not  yet  know  just  what  a  vitamine  is  or 
whether  it  is  an  amine  at  all  but  no  one  since  1911  has  been  able 
to  get  any  nearer  to  the  identification  than  Funk  and  while  he  has 
added  much  data  to  his  earlier  studies  he  has  himself  not  yet 
given  us  the  pure  vitamine.     For  that  reason  it  has  been  sug- 
gested by  various  people  that  the  name  vitamine  should  not  be 
used  since  it  has  no  sufficient  evidence  to  support  it.     Hopkins 
of  England  had  suggested  the  name  "  accessory  food  factors." 
E.  V.  McCollum  holds  that  we  should  call  them  the  "unidenti- 
fied dietary  factors"  and  added  later  to  this  phrase,  the  terms 
water-soluble  "B"  and  fat-soluble  "A"  after  the  fat  soluble  form 
was  discovered.     Most  chemists  feel,  however,  that  the  purpose 
of  nomenclature  is  brevity  combined  with  ready  recognition  of 
what  you  are  discussing  and  that  it  is  unnecessary  to  change  the  name 
vitamine  until  we  know  exactly  what  the  substances  are.     The 
result  is  that  while  still  a  mystery  chemically  they  remain  under 
the  name  of  vitamine  and  the  kinds  are  distinguished  by  the 
McCollum  terms  "fat-soluble  "A,"  water-soluble  "B,"  and  "C." 
We  see  that  beri-beri  then  was  responsible  for  Funk's  adding 
to  our  chemical  entities  a  new  member  but  it  does  not  yet  appear 
why  this  entity  concerns  our  normal  nutrition.     To  get  this  rela- 
tion we  must  turn  for  a  moment  to  the  state  of  knowledge  in 
1911  in  regard  to  foods  and  their  evaluation  and  what  was  going 
on  in  this  field  of  study  at  the  time. 


HOW  VITAMINES   WERE    DISCOVERED  11 

A  great  advance  in  measuring  food  value  was  the  discovery  of 
the  isodynamic  law.  Translated  into  ordinary  language  this 
law  states  that  when  a  person  eats  a  given  amount  of  a  given 
kind  of  food,  that  food  may  liberate  in  the  body  practically  the 
same  amount  of  energy  that  it  would  produce  if  it  were  burned  in 
oxygen  outside  of  the  body.  The  confirmation  of  this  law  pcr-~ 
mitted  us  to  apply  to  the  measurement  of  food  the  same  method 
we  had  already  learned  to  use  in  measuring  coal.  For  con- 
venience the  physicists  devised  a  heat  measure  unit  for  this  pur- 
pose and  naturally  called  it  by  a  word  that  means  heat,  namely, 
"  calorie."  Using  this  unit  and  applying  the  isodynamic  law  it 
was  merely  necessary  to  determine  two  things;  first,  how  many 
calories  a  man  produces  in  any  given  kind  of  work,  second  how 
many  calories  a  given  weight  of  each  kind  of  food  will  yield,  and 
then  give  the  man  as  many  calories  of  food  as  he  needs  to  meet 
his  requirements  when  engaged  in  a  given  kind  of  labor.  The 
measurement  and  tabulation  of  food  values  in  terms  of  calories  and 
the  investigation  of  the  calorie  needs  of  men  and  women  in  various 
occupations  has  been  one  of  the  great  contributions  of  the  past 
twenty  years  of  nutritional  study  and  to  the  progress  made  we 
owe  our  power  to  produce  proper  rations  for  every  type  of  worker. 
Army  rations  for  example  are  built  up  of  foods  that  will  yield 
enough  calories  to  supply  the  needs  of  a  soldier  and  during  the 
recent  war  extended  studies  conducted  in  training  camps  all  over 
the  United  States  have  shown  that  when  the  soldier  eats  all  he 
wants  he  will  consume  on  the  average  about  3600  calories  per 
day.  In  France  the  American  soldier's  ration  was  big  enough  to 
yield  him  4200  calories  per  day  if  he  ate  his  entire  daily  allowance. 

But  calories  are  not  the  only  necessities.  A  pound  of  pure 
fat  will  yield  all  the  calories  a  soldier  needs  in  a  day  but  his  lan- 
guage and  morals  wouldn't  stand  the  strain  of  such  a  diet. 
Neither  would  his  health,  for  not  only  does  his  body  demand  fuel 
but  also  that  it  be  of  a  special  kind.  While  there  are  many 
kinds  of  foodstuffs,  chemical  analysis  shows  that  they  are  mainly 
combinations  of  pure  compounds  of  relatively  few  varieties. 
The  chemists  call  these  proteins,  fats,  carbohydrates,  and  salts. 
Meats,  eggs,  the  curd  of  milk,  etc.,  are  the  principal  sources  of 
protein.  Sugars  and  starches  are  grouped  together  under  the 


12  VITAMINE   MANUAL 

name  of  carbohydrate.  By  salts  is  meant  mineral  matters  such 
as  common  salt,  iron  and  phosphorus  compounds,  etc.  In  select- 
ing foods  it  was  found  that  the  body  required  that  the  propor- 
tions of  these  four  substances  be  kept  within  definite  limits  or 
there  was  trouble.  We  know  now  that  a  man  can  get  along 
nicely  if  he  eats  50  grams  of  protein  per  day  and  makes  up  the 
rest  of  his  calories  in  carbohydrates  and  fats,  provided  that  to 
this  is  added  certain  requirements  in  salts  and  water. 

It  is  also  obvious  that  the  foods  given  must  be  digestible  and 
palatable. 

We  had  reached  this  status  some  time  before  1911.  But,  a 
short  time  before  this,  there  had  arisen  a  controversy  as  to  the 
relative  value  of  different  types  of  proteins.  The  animal-  vs. 
vegetable-protein  controversy  was  one  of  the  side  shows  of  this 
affair.  This  controversy  had  led  to  a  careful  study  of  the  differ- 
ent lands  of  proteins  that  are  found  in  foodstuffs.  Through  a 
brilliant  series  of  chemical  investigations  for  whose  description 
we  haven't  time  or  space  here,  chemists  had  shown  that  every 
protein  was  built  up  of  a  collection  of  acids  which  were  different 
in  structure  and  properties,  that  there  were  some  seventeen 
of  these  in  all  and  that  any  given  protein  might  have  present  all 
seventeen  or  be  lacking  in  one  or  more  and  that  the  propor- 
tions present  varied  for  every  type  of  protein.  It  was  then 
obvious  that  proteins  could  not  be  considered  as  identities.  More 
than  that,  it  was  the  necessary  task  of  the  food  expert  to  separate 
all  proteins  into  their  acids  or  building  stones  and  not  only  show 
what  was  present  and  how  much  but  determine  the  role  each 
played  in  the  body.  To  this  task  many  set  their  faces  and  hands. 

From  the  results  there  has  accrued  much  progress  in  the  evalua- 
tion of  proteins  but  an  unexpected  development  was  the  part 
played  by  these  investigations  in  the  story  of  the  vitamines. 

About  1909-1910  Professors  Osborne  and  Mendel  under  a 
grant  from  the  Carnegie  Institution  began  a  detailed  investiga- 
tion into  the  value  of  purified  proteins  from  various  sources.  In 
their  experiments  they  used  the  white  rat  as  the  experimental 
animal  and  proceeded  to  feed  these  animals  a  mixture  consisting 
of  a  single  purified  protein  supplemented  with  the  proper  propor- 
tions of  fat  carbohydrate,  and  mineral  salts.  Since  the  food  fur- 


HOW  VITAMINES   WERE   DISCOVERED  13 

nished  was  composed  of  pure  nutrients  and  always  in  excess  of 
the  appetite  of  the  rat  the  necessary  number  of  calories  was  also 
present.  These  researches  were  published  as  a  bulletin  (No. 
156)  by  the  Carnegie  Institution  in  1911,  the  same  year  that  Funk 
announced  his  Vitamine  discoveries.  It  was  timely  in  this  re- 
spect for  one  of  Osborne  and  Mendel's  discoveries  was  that  no 
matter  how  efficient  the  mixture  in  all  the  requirements  then 
known  to  the  nutrition  expert,  the  rats  failed  to  grow  unless  there 
was  added  to  the  diet  a  factor  which  they  found  in  milk.  In 
searching  for  this  factor  they  made  a  still  further  discovery  for 
on  fractioning  the  milk  they  soon  learned  that  the  unknown 
factor  was  distributed  in  two  different  parts  of  the  milk,  namely 
in  the  butter  fat  and  in  the  protein  free  and  fat-free  whey.  The 
absence  of  either  milk  fraction  was  sufficient  to  prevent  growth. 
The  1911  publication  merely  described  these  results  without 
attempting  to  explain  the  nature  of  the  growth  producing  factors 
but  the  vitamine  hypothesis  of  Funk  naturally  suggested  to  these 
authors  that  their  two  unknown  factors  might  be  similar  in  nature 
to  his  beri-beri  curative  factor  and  their  announcement  may  be 
justly  considered  a  point  of  junction  of  nutrition  theories  with 
the  vitamine  hypothesis. 

The  peculiarity  of  butter  fat  as  a  growth  stimulus  had  been 
considered  from  another  angle  by  a  German  worker,  Stepp.  In 
1909  this  student  of  nutrition  had  tried  to  estimate  the  importance 
of  various  types  of  fats  in  the  same  way  that  was  later  done  with 
proteins,  to  determine  whether,  like  proteins,  the  quality  of  the 
fats  varied  in  nutritive  efficiency.  His  experiments  were  also 
conducted  with  white  rats  and  the  main  outlines  of  his  methods 
and  observations  were  as  follows:  Rats  fed  on  a  bread  and  milk 
diet  grew  normally.  If  now  the  bread  and  milk  mixture  was 
extracted  with  alcohol-ether  the  residue  was  found  to  be  in- 
adequate for  growth  or  maintenance.  Stepp  assumed  that  this 
failure  could  naturally  be  ascribed  to  the  removal  of  the  fat  by 
the  alcohol-ether  mixture.  To  determine  the  efficiency  of  differ- 
ent kinds  of  fats  he  then  proceeded  to  substitute  in  combination 
with  the  alcohol-ether  extracted  diet  amounts  of  purified  fats 
corresponding  to  what  was  removed  by  the  alcohol-ether.  The 
results  were  totally  unexpected  for  none  of  the  purified  fats 


14  VITAMINE   MANUAL 

substituted  were  adequate  to  secure  growth!  When,  however, 
he  evaporated  off  his  alcohol-ether  from  the  extract  of  the  bread 
and  milk  and  returned  that  residue  to  the  diet,  growth  was  re- 
sumed as  before.  The  conclusion  was  obvious,  viz.,  that  alcohol- 
ether  takes  out  of  a  mixture  of  bread  and  milk  some  factor  that 
is  necessary  to  growth  and  that  factor  is  not  fat  but  something 
removed  by  the  extraction  with  the  fat.  These  results  led  Stepp 
to  suspect  the  existence  of  an  unidentified  factor  but  he  was  un- 
able to  identify  it  as  a  lipoid.  He  makes  the  following  state- 
ment which  is  now  significant:  "It  is  not  impossible  that  the  un- 
known substance  indispensable  to  life  goes  into  solution  in  the 
fats  and  that  the  latter  thereby  become  what  may  be  termed 
carriers  for  these  substances."  These  studies  were  published 
between  the  years  1909  and  1912  and  were  therefore  concurrent 
with  those  of  Funk  and  Osborne  and  Mendel. 

But  there  was  still  another  set  of  studies  that  led  up  to  this 
vitamine  work.  In  1907  E.  V.  McCollum  began  the  study  of 
nutrition  problems  at  the  Wisconsin  Experiment  Station.  At 
the  time  he  was  especially  interested  in  two  papers  that  had  been 
published  just  previous  to  his  entrance  into  the  problem.  One  of 
these  papers  by  Henriques  and  Hansen  told  how  the  authors  had 
attempted  to  nourish  animals  whose  growth  was  already  complete 
on  a  mixture  consisting  of  purified  gliadin  (the  principal  protein 
from  the  quantity  viewpoint  in  wheat),  carbohydrates,  fats,  and 
mineral  salts.  In  spite  of  the  fact  that  the  nitrogen  of  this  mixture 
was  sufficient  to  supply  the  body  needs,  as  proved  by  analysis 
of  the  excreta,  the  animals  steadily  declined  in  weight  from  the 
time  they  were  confined  to  this  diet.  The  authors  had  assumed 
that  the  gliadin  was  deficient  in  a  substance  necessary  to  growth 
(lysine)  but  since  their  studies  were  begun  only  after  the  animals 
had  reached  maximum  growth  they  expected  that  the  growth 
factor  would  not  be  necessary.  Why  had  their  animals  declined 
in  weight? 

The  second  paper  that  interested  McCollum  was  by  Wilcock 
and  Hopkins.  These  authors  carried  out  experiments  similar 
to  those  of  the  paper  just  cited  but  using  corn  protein  (zein)  in 
place  of  gliadin.  This  protein  had  already  been  shown  to  be 
deficient  in  a  chemical  constituent  known  as  tryptophan.  An- 


HOW  VIT AMINES   WERE   DISCOVERED  15 

imals  fed  on  the  zein  mixture  died  in  a  few  days  but  the  inex- 
plicable thing  was  that  when  the  missing  tryptophan  was  added 
to  the  diet  the  animals  lived  a  little  longer  but  finally  declined 
and  died.  Why? 

McCollum  wished  to  answer  this  "Why?"  These  experimenters 
had  complied  with  every  known  law  of  nutrition  and  yet  their 
mixtures  failed  to  nourish  the  animals.  What  was  lacking? 
Earlier  work  at  the  Station  by  Professor  Babcock  suggested  an 
interesting  line  of  attack  and  in  collaboration  with  Professors 
Hart  and  Humphries,  McCollum  began  a  series  of  studies  that 
have  become  classic  contributions  to  the  vitamine  hypothesis 
and  brought  this  worker  into  the  field  as  one  of  the  most  important 
contributors  to  the  subject.  His  initial  experiments  may  be 
briefly  summarized  as  follows:  Young  heifer  calves  weighing  350 
pounds  at  the  start  and  as  nearly  alike  in  size  and  vigor  as  could 
be  obtained  were  selected  as  experimental  animals.  These  were 
divided  into  groups  and  fed  with  rations  so  made  up  as  to  be  alike 
in  so  far  as  chemical  analysis  could  determine,  but  differing  in 
that  the  sources  of  the  ration  were  divided  between  three  plants. 
One  group  was  supplied  with  a  ration  obtained  entirely  from  the 
wheat  plant.  A  second  group  derived  their  ration  solely  from  the 
corn  plant.  A  third  from  the  oat  plant  and  a  fourth  or  control 
group  from  a  mixture  of  oat,  wheat  and  corn.  By  chemical 
analysis  each  group  received  enough  of  its  particular  plant  to 
produce  exactly  the  same  amount  of  protein,  fat  and  carbohydrate 
and  all  were  allowed  to  eat  freely  of  salt.  All  groups  ate  practi- 
cally the  same  amount  of  feed,  and  digestion  tests  showed  that 
there  was  no  difference  in  the  digestibility  of  the  different  rations. 
Exercise  was  provided  by  allowing  them  the  run  of  a  yard  free  of 
all  vegetation.  It  was  a  year  or  more  before  any  distinct  change 
appeared  in  the  different  groups.  At  that  time  the  cornfed 
animals  were  in  fine  condition.  On  the  contrary,  the  wheat-fed 
group  were  rough  coated,  gaunt  in  appearance  and  small  of  girth. 
The  oat-fed  group  were  better  off  than  the  wheat-fed  but  not  in 
so  good  shape  as  the  corn-fed.  In  reproduction  the  corn-fed 
animals  carried  their  young  well.  They  were  carried  for  the  full 
term  and  the  young  after  birth  were  well  formed  and  vigorous. 
The  wheat-fed  mothers  gave  birth  to  young  from  three  to  five 


16  V1T  \L 

end  of  the  normal  term.  The  young  wore  either 
born  dead  or  died  within  a  few  hours  after  birth.  All  were  much 
underweight.  The  oat-fed  mothers  produced  their  young  about 
two  weeks  before  the  normal  period.  Of  four  m.  one 

was  born  dead,  two  so  weak  that  they  died  within  a  day  or  two 
and  the  fourth  was  only  saved  by  special  measures.  The  young 
of  the  oat-fed  mothers  were  of  nearly  the  same  size,  however, 
as  those  of  the  corn-fed  mothers.  After  the  first  reproduction 
period,  the  mothers  were  kept  on  this  diet  another  year  and  the 
following  year  repeated  the  same  process  with  identical  results. 
During  the  first  milk-producing  period  the  average  production 
per  day  was  24.03  pounds  per  day  for  the  corn-fed.  10. oS  pounds 
for  the  oat-fed,  and  8.04  pounds  for  the  wheat-fed.  During  the 
second  period  it.  was  2S.O,  30.1,  and  10.1  pounds  per  day  res: 
during  the  first  thirty  days. 

Every  chemical  means  was  now  employed  to  determine  the 
causes  of  these  differences  and  without  success  MeCollum  then 
decided  to  attempt  to  solve  the  problem  by  selecting  small  animals 
(the  rat  was  used)  and  experiment  with  mixtures  consisting  of 
purified  proteins  from  different  sources,  combined  with  fats. 
carbohydrates  and  mineral  salts  until  a  clue  was  obtained  to  the 
nature  of  the  deficiencies.  His  early  results  in  this  direction 
confirmed  the  resul:  a  r  investigators,  animals  lived  no  longer 

on  these  diets  than  when  allowed  to  fast.  What  was  missing? 
Up  to  1911  the  main  result  of  these  experiments  had  been  to  call 
attention  to  the  peculiar  deficiencies  of  cereals  and  especially 
in  mineral  salts,  but  without  unlocking  the  mystery. 

These  collateral  investigations  show  how  in  all  parts  of  this 
country  and  on  the  other  side  of  the  ocean  events  were  marching 
toward  the  same  goal.  The  year  1911  then  is  a  significant  epoch. 
for  from  this  time  the  various  independent  efforts  began  to  link 
up  and  the  next,  few  years  carried  us  far  toward  the  goal. 

In  1912  MeCollum  was  working  with  a  mixture  consisting  of 
^  per  cent,  purified  protein  in  the  form  of  milk  curd  or  casein. 
20  per  cent,  lactose  or  milk  sugar,  5  per  cent,  of  a  fat  and  a  salt 
mixture  made  up  to  imitate  the  s:-  :\t  of  milk.     The  re- 

mainder of  that  mixture  was  starch.  With  this  mixture  MeCollum 
found  that  growth  could  be  produced  if  the  fat  were  butter  fat 


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FIG.  1.  COMPOSITE  CHART  OP  McCoLLUM  AND  DAVIS  PUBLICATIONS 

I  (from  Journ.  Biol.  Chem.,  1913,  xv,  167).    This  chart  shows  the  effect 
in  period  III  of  the  addition  of  an  ether  extract  of  egg,  1  gram  being  given 
every  other  day.     The  diets  for  periods  I-IV  were  as  follows : 

Periods I  II  III            IV 

Salt  mixture 6  6  6           5 

Casein 18  18  18          18 

Lactose 20  0  00 

Dextrin 0  59  74          74 

Starch 31  0  00 

Agar-agar 5  2  2           2 

Egg  (see  above) 0  0  *           0 

*  1  gram  extract  every  other  day. 

II  and  III  (from  Journ.  Biol.  Chem.,  1915,  xxiii,  231).    These  charts  show 
the  effect  (II)  of  the  addition  of  as  little  as  2  per  cent  wheat  embryo  a» 
sufficient  to  secure  normal  growth  when  it  serves  as  a  supply  of  the  B 
vitamine.     Chart  III  shows  that  even  when  the  wheat  embryo  is  increased 
to  30  per  cent  it  is  inadequate  for  growth  unless  the  A  is  also  present.    The 
diets  were  as  follows: 

Chart  H    Chart  III 

Dextrin 69.3  52.8 

Salt  mixture 3.7  2.6 

Butter  fat 5.0  0.0 

Agar-agar 2.0  2.0 

Casein 18.0  12.6 

Wheat  embryo 2.0  30.0 

17 


18  VITAMINE   MANUAL 

but  not  if  it  were  olive  oil,  lard,  or  vegetable  oils  of  various  sorts. 
Carrying  out  the  lead  here  suggested  he  tried  egg  yolk  fats.  They 
proved  as  effective  as  butter  fat. 

These  results  linked  up  with  those  of  Stepp  and  Mendel  and 
showed  that  butter  fat  and  egg  yolk  fat  contained  a  growth  factor 
which  was  missing  in  other  fats.  McCollum  named  this  the 
"unidentified  dietary  factor  fat-soluble  A." 

In  the  same  year  F.  G.  Hopkins  in  England  announced  that  the 
addition  of  4  per  cent  of  milk  to  diets  consisting  of  purified  nu- 
trients would  convert  them  into  growth  producers.  This  was 
too  small  an  amount  to  admit  of  attributing  the  cause  to  milk 
proteins,  fats,  carbohydrates,  or  salts.  Hopkins  therefore  sug- 
gested the  existence  of  unknown  factors  in  milk  of  the  type 
to  which  he  had  earlier  given  the  name  "accessory  factors."  This 
work  has  recently  been  repeated  by  Osborne  and  Mendel  who  fail 
to  find  the  high  potency  in  milk  ascribed  to  it  by  Hopkins  but  the 
latter's  work,  at  that  time,  was  accepted  without  question  and 
became  the  impetus  to  important  discoveries. 

Mendel  and  Osborne  had  meanwhile  investigated  more  in  de- 
tail their  milk  fractions.  They  obtained  results  that  confirmed 
McCollum's  findings  for  butter  fat  but  in  addition  they  showed 
that  by  removing  all  the  fat  and  protein  from  milk  they  obtained 
a  residue  which  played  an  important  part  in  growth  stimulation 
and  that  this  factor  was  different  from  the  salts  present  in  the 
mixture.  This  specially  prepared  milk  residue  they  called  pro- 
tein-free milk. 

The  next  few  years  are  a  melting  pot  of  investigations.  They 
included  some  sharp  controversies  over  nomenclature  and  many 
apparently  contradictory  conclusions  based  on  what  we  now 
know  to  be  insufficient  data.  The  principal  outcome  was  the 
identification  of  the  yeast  and  rice  polishing  substance  with  the 
factor  carried  by  protein-free  milk.  On  the  basis  of  these  results 
Funk  put  forward  the  idea  that  McCollum's  butter-fat  and  egg- 
yolk  factor  was  merely  vitamine  which  clung  to  the  fats  as  an 
adulterant.  It  was  soon  shown,  however,  that  butter  fat  could 
be  obtained  that  was  absolutely  free  of  nitrogen  and  still  be  stimu- 
latory to  growth.  It  was  therefore  clear  that  whatever  the  factor 
present  it  could  not  be  the  Funk  vitamine.  From  out  of  the  smoke 


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FIG.  2.  COMPOSITE  CHART  OF  OSBORNE  AND  MENDEL  PUBLICATIONS 

These  four  charts  all  show  the  power  of  sources  of  the  A  vitamine  to 
bring  about  recovery  after  failure  on  diets  lacking  that  vitamine. 

I  (from  Journ.  Biol.  Chem.,  1913-14,  xvi,  423).     In  this  group  the  diet 
consisted  of  the  following  percents:  Protein,  18;  starch,  26;  protein  free 
milk,  28;  lard,  28.     In  the  part  of  the  periods  marked  butter,  18 per  cent 
of  butter  was  substituted  for  an  equal  amount  of  lard. 

II  (from  Jour.  Biol.  Chem.,  1913,  xv,  311).    Shows  recovery  on  addition 
of  butter  fat  to  a  diet  containing  all  the  nutrients  and  artificial  protein 
free  milk.    These  diets  contained  the  following  percents:  Protein,  18; 
lactose,  23.8;  starch,  26;  milk  salts,  4.2;  total  fats,  28. 

III  (from  Journ.  Biol.  Chem.,  1915,  xx,  379).     These  show  the  effect  of 
various  sources  of  vitamine  A  such  as  egg  fat,  butter  fat  and  oleomargarine. 
The  broken  line  parts  show  the  failure  of  laboratory  prepared  lard  to  better 
the  commercial  lard  of  the  basal  diet  and  the  crossed  lines  the  immediate 
effect  when  a  true  source  of  vitamine  A  was  added.     Basal  diet:  Protein, 
18,  protein  free  milk,  28;  starch,  24-29;  lard,  7-28;  other  fats,  0-18. 

IV  (from  Journ.  Biol.  Chem.,  1913-14,  xvii,  401).    This  chart  shows  the 
failure  of  almond  oil  as  a  source  of  vitamine  A  and  the  prompt  recovery 
when  butter  fat  or  cod-liver  oil  was  used.    Basal  diet :  Edestin,  18 ;  starch, 
28;  protein  free  milk,  28;  lard,  8;  almond  oil  or  butter  fat  or  cod-liver  oil,  18. 

19 


20  VITAMINE   MANUAL 

of  this  controversy  came  an  ultimate  explanation  that  was  veiy 
simple.  There  were  two  factors  instead  of  one.  McCollum 
did  not  discover  the  presence  of  the  Funk  vitamine  in  his  mixtures 
at  first  because  it  was  carried  by  the  lactose  and  he  did  not  know 
it.  Finally,  to  cut  a  long  story  very  short,  these  two  factors  or 
vitamines  were  both  found  to  be  essential  to  growth  and  in  the 
feeding  mixtures  that  had  been  used  were  distributed  as  follows: 

Vitamine  A  Vitamine  B  (Funk's  vitamine) 

Fat-soluble  Water-soluble 

Non-antineuritic  Antineuritic 

Present  in  butter  fat  and  egg-yolk      Present  in  protein-free  milk,  ordi- 
fat  nary  lactose,  yeast  and  rice  pol- 

ishings 

With  these  points  cleared  up  each  nutrition  investigator  re- 
turned to  an  analysis  of  his  food  mixtures  and  proceeded  to  the 
location  in  sources  of  the  various  factors.  The  years  1912-1918 
are  mainly  contributory  to  further  knowledge  of  the  properties 
of  these  two  vitamines,  their  reactions,  source,  behavior,  etc. 
In  1912,  however,  Hoist  and  Frohlich  began  a  study  of  scurvy 
that  was  to  culminate  later  by  adding  to  the  list  a  new  member 
of  the  family,  viz.,  vitamine  "C." 

The  disease  of  scurvy  and  its  prevention  by  use  of  orange  juice, 
potatoes,  etc.,  was  a  well  known  phenomenon  and  to  the  curative 
powers  of  lime  juice  we  owe  the  name  "lime-juicers"  as  a  synonym 
for  the  British  merchant  marine. 

Following  his  discovery  of  vitamine  as  the  preventative  sub- 
stance to  beri-beri,  Funk  had  outlined  a  theory  of  "avitaminoses" 
as  the  responsible  cause  of  several  other  types  of  diseases,  including 
scurvy,  rickets,  pellagra,  and  beri-beri.  In  other  words,  he  sug- 
gested that  the  etiology  of  these  diseases  would  be  found  to  lie 
in  the  lack  of  the  vitamine  factors.  His  views  at  the  time 
were  largely  hypothetical  since  the  only  one  of  his  avitaminoses 
then  demonstrated  was  beri-beri,  but  the  hypothesis  attracted 
attention  and  developed  a  new  method  of  study  as  it  had  in  matters 
of  normal  nutrition. 

Between  1907  and  1912  Hoist  and  Frohlich  had  made  exhaus- 
tive studies  of  the  causes  of  scurvy  and  had  reached  the  conclu- 
sion that  its  cause  was  due  to  the  absence  of  some  factor,  admittedly 


HOW  VITAMINES   WERE   DISCOVERED  21 

unknown,  but  as  strongly  indicated  as  in  the  case  of  beri-beri. 
Hoist  pointed  out  that  a  guinea  pig  restricted  to  a  diet  of  oats 
became  affected  with  scurvy.  McCollum  as  well  as  others  were 
attracted  to  this  problem  and  in  1918  McCollum  stated  that 
scurvy  was  not  due  to  a  lack  of  a  dietary  factor  but  to  the  absorpt 
tion  from  the  intestine  of  the  poisonous  products  resulting  from 
abnormal  decomposition  of  the  food  and  especially  of  protein 
food.  He  studied  the  guinea  pig  on  an  oat  diet  and  drew  the  con- 
clusion that  while  it  does  induce  scurvy  this  result  is  not  due  to 
the  absence  of  any  specific  factor  in  the  oat  diet.  He  showed  that 
while  the  oat  kernel  contains  all  the  chemical  elements  and  com- 
plexes necessary  for  the  growth  and  health  of  an  animal  these 
elements  are  not  in  suitable  proportions.  It  lacks  certain  mineral 
salts  and  its  content  of  the  "A."  vitamine  is  too  low  to  permit 
oats  alone  to  give  satisfactory  growth  results.  Furthermore  its 
proteins  are  not  of  as  good  quality  as  those  of  milk,  eggs,  and 
meat.  By  merely  supplementing  the  oat  diet  with  better  protein, 
salts,  and  a  growth  promoting  fat,  he  reported  that  a  guinea  pig 
could  be  developed  normally  without  further  addition  and  that 
therefore  it  was  impossible  to  show  that  any  unknown  factor  was 
responsible  for  the  scurvy  symptoms.  McCollum  also  reported 
that  the  guinea  pig  could  develop  scurvy  even  when  his  diet  was 
supplemented  with  fresh  milk  and  since  milk  was  a  complete 
food  it  followed  that  the  cause  of  the  disease  must  be  sought  out- 
side of  dietary  factors. 

Examination  of  guinea  pigs  that  died  of  scurvy  showed  that 
the  cecum  was  always  full  of  putrefying  feces.  This  obser- 
vation suggested  that  the  mechanical  difficulty  these  animals 
have  in  removing  feces  from  this  part  of  the  digestive  tract 
might  have  something  to  do  with  the  disease.  McCollum  and  his 
workers  were  confirmed  in  their  views  by  the  excellent  results 
that  followed  the  use  of  a  mineral  oil  as  a  laxative.  Another 
piece  of  evidence  they  gave  for  their  views  was  that  when 
animals  were  fed  on  oats  and  milk  the  onset  of  the  scurvy  could 
be  delayed  by  merely  adding  the  cathartic,  phenolphthalein,  to 
the  mixture.  They  met  the  argument  of  the  curative  power 
of  orange  juice  by  preparing  an  artificial  juice  of  citric  acid, 
inorganic  salts  and  cane  sugar  and  showing  that  this  synthetic 


22  VITAMINE   MANUAL 

mixture  which  held  only  known  substances  was  capable  of 
protecting  aninals  from  scurvy  over  a  long  period  of  time.  With- 
out going  further  into  the  evidence  presented  by  these  workers 
McCollum  was  sufficiently  convinced  of  the  correctness  of  his 
own  views  to  not  only  state  them  in  his  researches  but  to  set  them 
forth  at  length  for  public  information  in  his  book  entitled  The 
Newer  Knowledge  of  Nutrition.  In  spite  of  all  this  evidence  his 
views  failed  to  convince  the  holders  of  the  vitamine  hypothesis. 
Harden  and  Zilva  and  Chick  and  Hume  in  England  freely  criti- 
cised his  conclusions  because  whole  milk  was  used  in  his  experi- 
ments and  no  attention  paid  to  the  amounts  eaten.  It  was  then 
well  known  that  if  enough  whole  milk  is  eaten  scurvy  will  not  de- 
velop. Cohen  and  Mendel  autopsied  normal  guinea  pigs  and 
found  that  the  cecum  was  nearly  always  full  of  feces.  On  the 
other  hand  in  autopsies  of  many  pigs  dead  from  scurvy  only 
one-fourth  were  found  to  show  the  impaction  of  feces  claimed 
by  McCollum  as  cause  of  the  disease.  Milk  is  constipating  to 
guinea  pigs.  Large  amounts  of  milk  should  therefore  have  in- 
creased scurvy  if  the  cause  stated  by  McCollum  was  the  real 
one.  On  the  contrary  large  amounts  of  milk  prevented  scurvy 
and  small  doses  permitted  it  to  develop.  The  use  of  coarse 
materials  as  a  preventative  of  constipation  failed  to  prevent 
scurvy  onset.  Hess  and  linger  found  that  cod-liver  oil  and  liquid 
petrolatum  prevented  constipation  but  failed  to  prevent  scurvy. 
The  attack  on  the  McCollum  view  continued  from  various 
quarters.  Chick  and  Hume  in  England  examined  his  grain  and  milk 
fed  series  and  showed  that  those  receiving  much  milk  and  little  grain 
recovered  while  those  on  the  reverse  diet  died.  They  held  that 
all  guinea  pigs  with  scurvy  become  constipated  regardless  of  the 
diet.  They  gave  large  quantities  of  dried  vegetables  well  cooked 
in  water,  in  order  to  provide  bulk,  but  this  did  not  prevent  scurvy 
and  neither  did  the  use  of  mineral  oil.  Hess  found  that  in  in- 
fants with  scurvy  there  is  a  history  of  constipation  but  that  while 
potatoes  which  are  not  laxative  cure  scurvy,  malt  soups  which  are 
laxative  permit  its  development.  He  found  that  scurvy  in  in- 
fants is  relieved  by  amounts  of  orange  juice  entirely  too  small 
to  have  a  marked  laxative  action  and  was  unable  to  secure  cures 
with  McCollum's  artificial  orange  juice.  The  most  convincing 


HOW  VITAMINES  WEBE   DISCOVERED  23 

argument  was  the  discovery  that  orange  juice  adminstered  in- 
travenously still  exerted  a  curative  action  which  could  not  in 
any  way  be  laid  to  its  effect  on  constipation. 

To  these  attacks  McCollum's  co-worker,  Pitz,  suggested  a 
new  hypothesis.  It  was  well  known  that  in  rats  and  man  the  in- 
testinal flora  can  be  changed  from  a  putrefactive  form  to  a  non- 
putrefactive  type  by  feeding  milk  sugar  or  lactose.  If  this  were 
true,  as  was  admitted  by  all,  and  the  scurvy  due  to  the  absorption 
of  putrefactive  products,  this  absorption  might  still  be  the  causal 
factor  whether  constipation  was  present  or  absent.  To  determine 
this  point  he  fed  his  guinea  pigs  on  oatmeal  to  which  he  added  a 
carbohydrate  diet.  When  the  carbohydrate  was  lactose  he  was 
able  to  cure  and  prevent  scurvy.  This  evidence  was  not  consid- 
ered convincing,  however,  since  in  his  experiments  milk  was  given 
freely.  Furthermore,  Cohen  and  Mendel  demonstrated  that  in 
their  experiments  pure  lactose  neither  prevented  nor  cured  scurvy 
while  Harden  and  Zilva  could  find  no  antiscorbutic  value  in  either 
cane  sugar,  fructose,  or  sirup.  These  authors  believed  and  stated 
that  Pitz's  results  were  entirely  attributable  to  the  free  use  of 
raw  milk. 

As  this  milk  factor  came  increasingly  to  the  attention  in  the 
controversy  it  was  natural  that  students  began  to  reexamine 
this  product  more  carefully.  The  vitamine  advocates  at  first 
believed  that  its  potency  as  an  antiscorbutic  was  of  course  due 
to  the  vitamines  already  found  present  therein,  viz.,  the  "A"  or 
the  "B."  But  there  began  to  be  difficulties  with  this  view.  Hess 
found  that  eggs  and  cod-liver  oil,  both  rich  in  "A"  were  of  no 
value  as  scurvy  cures.  These  experiments  eliminated  the  "A"  as 
the  curative  factor.  Cohen  and  Mendel  used  a  mixture  of  yeast 
and  butter  in  their  experiments  without  success.  These  exper- 
iments threw  doubt  on  the  "B"  as  a  curative  factor.  Studies  in 
heated  milk  had  also  shown  that  the  scurvy  curing  power  was 
destroyed  by  such  procedures  as  heating  and  that  pasteurized 
milk  was  not  as  good  as  raw  milk.  This  heating  on  the  other 
hand  did  not  destroy  the  antineuritic  power  of  the  milk  nor  its 
growth-stimulating  properties.  The  combined  result  of  all  these 
studies  was  to  eliminate  both  the  "A"  and  the  "B"  as  the  vita- 
mines  with  antiscorbutic  power  without  suggesting  a  better  hy- 
pothesis than  McCollum's. 


24  VITAMINE   MANUAL 

Gradually,  however,  it  became  evident  that  while  scurvy  is 
not  prevented  by  either  of  these  vitamines  Funk's  hypothesis 
and  Hoist  and  Frohlich's  experimental  evidence  was  correct  and 
McCollum's  view  wrong.  The  answer  lay  in  the  discovery  of  a 
third  vitamine,  water-soluble  like  "B"  but  otherwise  of  entirely 
different  behavior  and  properties.  J.  C.  Drummond  of  England 
finally  suggested  its  inclusion  in  the  family  and  the  name  water- 
soluble  "C."  As  soon  as  its  presence  was  admitted  and  its  prop- 
erties roughly  determined  the  way  was  opened  to  development 
of  the  antiscorbutic  vitamine  hypothesis  and  that  has  now  pro- 
ceeded as  rapidly  as  in  the  other  fields.  During  the  past  year 
many  contributions  have  been  made  in  this  field.  Sherman,  La 
Mer,  and  Campbell  have  recently  published  results  that  have 
taught  us  much  about  the  measurement  of  this  new  member 
and  its  manipulation  in  experimental  study  of  scurvy. 

The  year  1920,  then,  has  brought  us  to  a  recognition  of  at  least 
three  members  of  the  family.  Still  more  recently  another  de- 
ficiency disease  has  been  under  investigation  and  Hess  has  found 
in  cod-liver  oil  a  remedy  for  rickets  that  he  cannot  believe  owes 
its  efficiency  to  the  "A"  type.  Mellanby  of  England  believes  the 
"A"  vitamine  is  the  preventive  factor  in  this  disease  but  Hess's 
results  at  least  suggest  the  possibility  that  the  antirachitic  vi- 
tamine may  be  separate  and  distinct  from  any  of  those  yet  named, 
possibly  vitamine  "D?"  Others  are  beginning  to  doubt  the 
identity  of  the  rat  growth  promoter  and  the  beri-beri  curing 
complexes  and  feel  that  the  "B"  itself  may  be  the  name  of  a  group 
instead  of  a  single  entity.  All  of  these  features  make  one  feel 
uncertain  to  say  the  least,  as  to  the  limits  of  this  vitamine  family 
or  of  the  future  possibilities  but  enough  has  been  given  to  indicate 
the  historical  development  to  date  and  we  can  now  turn  to  more 
special  features  of  the  subject  and  their  bearing  on  every  day 
affairs. 


CHAPTER  II 

THE  ATTEMPTS  TO  DETERMINE  THE  CHEMICAL  NATURE  OF  A 

VITAMINE 

The  discovery  of  the  existence  of  an  unknown  substance  is 
naturally  a  stimulation  to  investigation  of  its  nature.  In  the 
case  of  the  vitamines  we  have  many  researches  to  this  end  but 
extremely  meagre  results.  We  are  today  actually  no  nearer  the 
goal  of  identification  than  we  were  in  1911  when  Funk  pub- 
lished his  studies  on  the  beri-beri  curing  type.  In  brief,  we  do 
not  know  what  a  vitamine  is.  Nevertheless,  it  will  be  of  interest 
to  the  student  to  review  the  attempts  that  have  been  made  to 
isolate  these  substances  for  such  attempts  must  furnish  the  start- 
ing point  for  further  studies  and  their  description  will  help  to 
make  clear  the  nature  of  the  problem  involved. 

The  most  extensive  investigations  have  dealt  with  the  first 
type  discovered,  namely  the  vitamine  "B"  or  Funk  antineuritic 
type.  In  1911  Cooper  and  Funk  found  that  the  alcoholic  extract 
of  rice  polishings  could  be  precipitated  with  phosphotungstic  acid 
and  that  this  procedure  permitted  them  to  obtain  a  fraction  that 
was  particularly  potent  and  free  from  proteins,  carbohydrates, 
and  phosphorus.  Funk  carried  this  investigation  farther  and 
fractioned  the  phosphotungstic  acid  precipitate  with  silver  nitrate, 
following  the  usual  procedure  for  separating  nitrogenous  bases. 
From  the  silver-nitrate  baryta  fraction  he  obtained  a  crystalline 
complex  melting  at  233°C.  to  which  he  gave  the  formula 
Ci7H2oO7N2.  This  substance  was  curative  for  pigeons  and  the 
fractioning  process  was  applied  by  him  to  yeast  and  other  food- 
stuffs with  similar  results.  From  these  results  Funk  believed  the 
vitamine  to  belong  to  a  class  of  substances  known  as  the  pyri- 
midine  bases.  Later,  when  working  with  Drummond,  Funk  was 
forced  to  admit  that  his  crystalline  complex  was  not  the  pure 
substance,  as  analysis  showed  that  it  contained  large  amounts 
of  nicotinic  acid.  His  product  might  well  be  considered  as 
nicotinic  acid  contaminated  with  vitamines. 

25 


26  VITAMINE   MANUAL 

Suzuki,  Shimamura  and  Odake  also  used  the  phosphotungstic 
precipitation  method  and  claimed  to  have  prepared  the  crystalline 
antineuritic  substance  which  they  called  oryzanin  in  the  form  of 
a  crystalline  picrate.  Drummond  and  Funk  repeated  this  work, 
but  were  unable  to  confirm  the  Japanese  results.  A  group  of 
British  chemists  (Edie,  Evans,  Moore,  Simpson  and  Webster) 
obtained  an  active  fraction  from  yeast  and  succeeded  in  separating 
this  into  a  crystalline  basic  member  belonging  to  the  pyrimidine 
group  which  they  called  torulin. 

None  of  these  three  preparations  have  stood  the  test  of  analysis 
however  and  their  curative  properties  seem  to  lie  in  their  greater 
or  less  contamination  with  the  actual  substance,  whatever  it  is. 
Numerous  modifications  of  the  fundamental  method  for  extracting 
the  substance  have  been  planned  and  executed.  Funk  for  example 
has  shown  that  if  the  phosphotungstic  precipitate  is  treated  with 
acetone  it  is  possible  to  separate  it  into  an  acetone  soluble  and  an 
acetone-insoluble  fraction  and  that  the  curative  fraction  is  in  the 
latter.  McCollum  has  reported  that  while  ether,  benzene  and 
acetone  cannot  be  used  to  extract  the  B  vitamine  from  its  source, 
benzene,  (and  to  a  slight  extent  acetone)  will  dissolve  the  vitamine 
if  it  is  first  deposited  from  an  alcohol  extract  on  dextrin.  These 
observations  have  not  yielded  any  further  clew  to  the  nature  of 
the  substance. 

Recently  Osborne  and  Wakeman  have  proposed  a  modification 
which  yields  a  concentrate  of  high  potency.  Their  method  is  to 
add  fresh  yeast  to  slightly  acidified  boiling  water  and  continue  the 
boiling  for  about  five  minutes.  This  process  coagulates  the  pro- 
teins that  are  present  and  permits  their  removal  by  filtration. 
The  protein-free  filtrate  appears  to  contain  all  of  the  vitamine 
originally  present  in  the  yeast  but  attempts  to  precipitate  the 
vitamine  fractionally  from  the  evaporated  filtrate  by  means  of 
increasing  concentration  of  added  alcohol  has  been  only  partially 
successful.  The  method  however  yields  a  concentrated  extract 
and  Harris  has  made  use  of  this  process  to  prepare  tablets  for 
medicinal  purposes. 

Seidell  and  Williams  some  time  ago  devised  a  procedure  which 
seemed  to  give  promise  of  good  results.  Their  discovery  was  that 
when  a  filtrate  from  autolysed  yeast  is  prepared,  rich  in  the 


CHEMICAL  NATURE   OF  A  VITAMINE  27 

vitamine,  and  is  shaken  with  a  specially  activated  fuller's  earth 
(the  preparation  produced  by  Lloyd  and  known  as  Lloyd's  reagent 
has  this  power)  in  a  proportion  of  50  grams  to  the  liter  of  extract 
the  vitamine  is  absorbed  by  the  earth  and  when  the  latter  is 
filtered  off  it  carries  the  vitamine  with  it.  In  their  process  they 
shake  the  mixture  for  about  one-half  hour  and  then  remove  the 
earth  by  filtration.  Analysis  of  the  yeast  liquor  after  the  extrac- 
tion shows  it  to  contain  practically  the  same  solids  as  originally 
present  but  to  have  lost  practically  all  its  vitamine.  The  latter 
is  firmly  attached  to  the  earth  and  repeated  washing  with  water 
fails  to  remove  any  appreciable  amount  of  vitamine  from  it. 
Furthermore  the  vitamine-activated  fuller's  earth  retains  its 
active  vitamine  properties  for  at  least  a  period  of  two  years. 
Large  amounts  of  the  vitamine  can  be  accumulated  in  this  way 
and  when  fed  to  animals  or  infants  the  vitamine  is  liberated 
physiologically  and  produces  the  usual  effects  of  a  vitamine 
extract.  When  this  discovery  was  made  the  discoverers  thought 
that  in  the  fuller's  earth  they  had  a  means  for  arriving  at  the 
identification  of  the  substance  but  attempts  to  recover  the  vita- 
mine  from  the  earth  developed  unexpected  difficulties.  Acids 
were  found  to  split  it  off  but  they  also  split  off  aluminium 
compounds  and  left  an  impure  mixture  little  better  than  the 
original  extract  for  study.  By  using  a  dilute  alkali  they  were  able 
to  obtain  the  substance  without  aluminium  contaminations  and 
by  this  method  they  actually  obtained  some  microscopic  fibrous 
needles  which  were  curative.  These  needles  however  on  recrystal- 
lization  resulted  in  the  production  of  a  compound  contaminated 
with  adenin  or  rather  in  adenin  contaminated  with  the  curative 
substance  and  on  standing  for  some  time  the  adenin  crystals 
gradually  lost  their  curative  power.  These  results  led  Williams 
to  suggest  an  interesting  hypothesis.  By  experiments  conducted 
with  the  hydroxy-pyridines  he  believed  that  he  had  demonstrated 
a  relation  between  tautomerism  or  changed  space  relations  in 
these  sort  of  substances  and  curative  properties.  He  states  his 
view  as  follows: 

The  vitamines  contain  one  or  more  groups  of  atoms  constituting  nuclei 
in  which  the  curative  properties  are  resident.  In  a  free  state  these  nuclei 
possess  the  vitamine  activity  but  under  ordinary  conditions  are  spontane- 


->  VITAMIXE   MANUAL 

oushr  transformed  into  isomers  which  do  not  possess  an  antineuritic  power. 
Tfce  complementary  substances  or  substituent  groups  with  which  these 
nuclei  are  more  or  less  firmly  combined  in  nature  exert  a  stabilizing  and 
perhaps  otherwise  favorable  influence  on  the  curative  nucleus,  but  do  not 
I  h<  mil  nil  i  i  possess  tile  vitamine  type  of  physiological  potency.  Accord- 
ingly H  is  believed  that  while  partial  cleavage  of  the  vitamines  may  result 
only  in  a  modification  of  their  physiological  properties,  by  certain  means 
so  far  as  to  effect  a  complete  separation  of  nucleus  and 
r,  and  if  it  does  so  will  be  followed  by  a  loss  of  curative  power  due 
to  isomerism.  The  basis  for  the  assumption  that  an  isomeriiation  con- 
stitutes the  final  and  physiologically  most  significant  step  in  the 
inactivation  of  a  vitamine  is  found  in  the  studies  of  synthetic  antineuritic 
products.  This  assumption  is  supported  by  evidence  ....  of  the 
existence  of  such  isomerism  in  the  crystalline  antineuritic  substances 
obtainable  from  brewer's  yeast. 

According  to  this  view  the  active  adenin  obtained  was  not  a 
contamination  but  an  inactive  isomer  of  the  active  substance. 
The  hydroxy-betaines  which  Williams  prepared  in  defense  of 
his  theory  have  been  repeatedly  tested  but  have  in  general  failed 
to  confirm  his  view  which  stands  today  as  an  interesting  sugges- 
tion but  without  confirmatory  evidence.  Other  attempts  by 
these  authors  to  fraction  their  alkaline  extract  of  fuller's  earth 
have  been  unsuccessful.  It  is  of  course  well  known  that  alkali 
acts  upon  the  vitamine  destructively.  On  this  account  the  authors 
of  this  method  operate  as  rapidly  as  possible  and  restore  the  alkali 
extract  to  a  neutral  or  acid  medium  quickly.  The  aqueous  extract 
obtained  from  the  earth  in  this  manner  has  been  shown  by  Seidell 
to  possess  only  about  one-half  of  the  vitamine  originally  present 
in  the  solid  but  the  vitamine  in  it  is  shown  to  be  fairly  stable. 
Seidell  has  not  yet  determined  how  long  it  remains  so.  Attempts 
to  recover  the  vitamine  from  such  aqueous  solutions  have  however 
totally  failed  to  date.  To  quote  Seidell  from  a  recent  pub- 
lication: 

By  careful  evaporation  of  the  solution  the  products  successively  obtained 
•hna  more  or  less  activity  by  physiological  tests  but  in  no  case  does  the 
the  appearance  or  character  which  a  pure  prod- 
be  expected  to  show.  Solvents  such  aa  benzene,  ethylacetate 
fail  to  effect  a  separation  of  active  from  inactive  material. 
tamrae  tends  to  distribute  itself  between 
the  fractions  rather  than  to  become  concentrated  in  one  or  the  other. 


CHEMICAL  3TATUKE  OF  A  VTTAMDTE  29 

The  difficulties  encountered  by  Seidefl  in  this  fractioning  study 
hare  fed  him  to  adopt  Wabcbe's  idea  thai  vitamines  are  of  the 
nature  of  enzymes  and  hence  present  afl  the  difficulties  of  identi- 
fication and  isolation  of  those  substance*, 

Daring  1920  Myers  and  VoegtKn  attacked  the  problem.  They 
havemadeadiscoYeTythatisiigefalasagcpaTatM>pioft».  Tins 
is  thai  the  "B"  vitamine  is  not  only  soluble  in  water,  bat  also 
in  ofive  ofl  and  in  olele  acid.  By  shaking  an  autolysed  yeast 
extract  with  these  solvents  in  the  proportion  of  1  cc.  of  sohrent  to 
each  4  cc.  of  extract  the  vitamine  passes  into  the  ofl.  When  this 
activated  ofl  is  filtered  and  taken  up  with  eight  to  ten  volumes  of 
ether  it  is  possible  to  concentrate  the  ether  extract  in  vaeuo  and 
toextnurtfromftwith0.1i>ereent  HQan  Aside 

from  this  observation  however  nothing  further  has  been  reported 
and  the  possibility  of  this  method  of  concentration  remains  yet 
to  be  exploited.  They  did  report  other  methods  of  fraetkxnng 
which  yielded  crystals  but  failed  to  produce  a  pure  active  sub- 
stance. Tliese  tumults  add  nothing  to  what  has  been  previously 
reported  except  a  new  method  of  fractioning  and  the 
of  the  following  substances  as  contributing  nothing  to 
activity  (pannes,  hJstJdinp,  proteins  and  albumoses).  The  crys- 
tals they  obtained  were  contaminated  with  hJs4«mmp- 

The  World  War  has  prevented  fnfl  knowledge  of  the  work  of 
the  German  investigators  but  nothing  has  appeared  that  indicates 
any  progress  in  this  field  with  the  exception  of  a  paper  by  Aber- 


and  ocnauniann  ft  ml  HOfti^*  M^M  y  by  UonneaiHer.  111^ 
Aberhalden  paper  yields  no  new  data  of  any  moment  and  no  active 
substances  in  pure  condition  are  reported-  The  reports  from 
Hofmeister  are  to  the  effect  that  he  has  isolated  a  verj 
solution  belonging  to  the  pyiimidme  series.  It  yields  a 
fane  hydrochloride  and  double  salt  with  gold  chloride 
given  it  the  formula  CsHnNO*. 

The  author  has  recently  been  able  to  obtain  a 
of  vitamine  from  an  extract  of  alfalfa  or  autoiysed  yeast  with 
the  aid  of  a  carbon  specially  activated  by  Me  Kee  of 
University  for  the  adsorption  of  basic  snbstanrp. 
has  been  found  quite  as  effective  as  the  fuller's  earth  and  it 
to  recover  the  vitamme  from  the  carbon  with 


30  VITAMIXE   MANUAL 

by  arid.  Glacial  acetic  and  heat  are  especially  favorable  for  this 
process.  The  study  of  this  concentrate  has  not,  however,  yet 
reached  a  stage  where  it  contributes  any  real  data  on  the  subject 
but  merely  provides  another  method  for  forming  concentrates. 

If  we  were  to  characterize  the  present  status  of  the  search  for 
the  "B"  type  it  might  be  said  to  have  resolved  itself  into  obtaining 
concentrates  of  high  potency  as  the  first  step  in  the  process  and 
this  type  of  investigation  is  now  going  on  in  many  laboratories. 

If  the  data  is  then  meagre  in  the  field  of  the  "B"  vitamine  it 
is  still  more  limited  in  the  case  of  the  "A"  and  the  "C."  One  of 
the  earliest  difficulties  encountered  in  the  study  of  the  "A"  vita- 
mine  was  the  failure  of  fat  solvents  to  extract  the  material  from 
its  richest  vegetable  sources.  If  butter  or  egg  yolk  is  extracted 
with  ether,  the  fat  obtained  is  rich  in  the  "A"  vitamine.  If,  how- 
ever, ether-extraction  is  applied  to  green  leaves  or  seeds  it  re- 
moves the  oils  but  these  oils  contain  little  or  no  vitamine.  Press- 
ing methods  also  fail  to  remove  the  substance  from  vegetable 
sources.  For  example,  if  we  press  or  extract  cotton  seed  we  ob- 
tain the  oil  but  the  vitamine  is  retained  in  the  press  cake.  Mc- 
Collum  suggested  the  following  explanation  for  this  behavior. 
His  idea  is  that  the  "A"  vitamine  while  soluble  in  fat  is  so  bound 
up  in  the  vegetable  source  that  extraction  methods  fail  to  loosen 
it.  When  these  vegetables  are  eaten  the  vitamine  is  set  free  in 
the  process  of  digestion  and  being  fat-soluble  passes  into  solution 
in  the  animal  fats.  Hence,  when  these  fats  contain  it  in  solution, 
they  retain  it  in  the  process  of  extraction  while,  lacking  this 
separatory  process,  ether  fails  to  loosen  it  from  the  vegetable 
binding.  Recently,  however,  Osborne  and  Mendel  have  pre- 
sented data  in  regard  to  this  binding  and  shown  that  if  for  ether 
we  substitute  an  ether-alcohol  mixture  the  removal  of  the  "A" 
with  the  fat  is  fairly  complete  even  from  vegetable  sources.  They 
advance  the  idea  that  preliminary  treatment  with  alcohol  is  a 
process  which  will  materially  assist  in  breaking  the  attachment 
of  the  vitamine  and  render  its  removal  with  the  fat  solvent  effec- 
tive. Butter-fat  rich  in  the  "A"  vitamine  has  been  conclusively 
shown  to  be  free  of  nitrogen  and  phosphorus  and  it  is  generally 
assumed  that  the  "A"  vitamine  is  a  nitrogen-free  and  phosphorus 
free  compound.  Further  than  that  however  we  know  nothing 
of  its  nature. 


CHEMICAL  NATURE  OF  A  YTTAMOfK  31 

Concerning  the  "C"  we  know  only  that  it  is  like  the  "B,"  water- 
soluble  and  we  know  somewhat  of  its  properties,  but  nothing  of  its 
chemical  nature. 

One  of  the  greatest  difficulties  still  encountered  in  the  study 
of  chemical  fractions  is  the  delay  in  identification  of  the  active 
portion.  For  this  purpose  we  must  rely  on  tests  that  are  far  from 
delicate  and  time-consuming  to  a  degree.  As  a  result  the  study 
of  only  a  few  fractions  must  extend  over  long  periods  of  time  with 
all  the  cumulation  of  difficulties  in  the  way  of  change  in  material, 
etc.  that  this  delay  implies.  An  idea  of  these  difficulties  can  best 
be  obtained  by  a  review  of  our  present  methods  for  vitamine 
testing  and  these  methods  constitute  the  subject  matter  of  the 
next  chapter. 


CHAPTER  III 
THE  METHODS  USED  ix  TESTING  FOR  VITAMINES 

It  will  be  evident  that  in  the  absence  of  exact  tests  for  a  sub- 
stance which  is  unknown  chemically  the  problem  of  detecting  its 
presence  must  be  a  matter  of  indirect  evidence.  When  a  chemist 
is  presented  with  a  solution  and  asked  to  determine  the  presence  or 
absence  of  lead  in  that  solution  he  knows  what  he  is  seeking,  what  its 
properties  are  and  how  to  proceed  to  not  only  determine  its  pres- 
ence but  to  measure  exactly  the  amount  present.  No  such  possi- 
bility is  present  in  a  test  for  vitamines,  but  this  lack  of  knowledge 
as  to  the  vitamine  structure  has  not  left  us  helpless.  We  do 
know  enough  of  its  action  to  permit  us  to  detect  its  presence  and 
the  technique  that  has  been  developed  for  this  purpose  is  now 
well  standardized  and  involves  no  mysteries  beyond  the  com- 
prehension of  the  layman.  In  the  present  chapter  is  outlined 
the  development  of  vitamine  testing  together  with  a  discussion 
of  some  of  the  deficiencies  and  the  problems  for  the  future  that 
these  deficiencies  suggest. 

When  Cassimir  Funk  made  his  original  studies  of  the  chemical 
fractions  of  an  alcohol  extract  of  rice  polishings  he  utilized  a 
discovery  of  the  Dutch  chemist  Eijkman.  We  have  already 
referred  to  this  disco  very,  viz.,  that  by  feeding  polished  rice  to 
fowls  or  pigeons  they  could  be  made  to  develop  a  polyneuritis 
which  is  identical  in  symptoms  and  in  response  to  the  curative 
action  of  vitamine,  to  the  beri-beri  disease.  A  normal  pigeon 
can  be  made  to  eat  enough  rice  normally  to  develop  the  disease 
in  about  three  weeks.  The  interval  can  be  somewhat  shortened 
by  forced  feeding.  As  soon  as  the  symptoms  develop  the  bird  is 
ready  to  serve  as  a  test  for  the  presence  or  absence  of  the  anti- 
neuritic  vitamine.  If  at  this  time  we  have  an  unknown  substance 
to  test  it  can  be  administered  by  pushing  down  the  throat  or 
mixed  with  the  food  or  an  extract  can  be  made  and  administered 
intravenously.  If  the  dose  is  curative,  the  bird  will  show  the  effect 
by  prompt  recovery  from  all  the  symptoms  of  the  disease  in  as  short 


TESTING    FOR   VITAMIXES 


33 


a  time  as  six  to  eight  hours.  Such  a  procedure  provides  a  quali- 
tative test  which  can  be  made  roughly  quantitative  by  varying  the 
dosage  until  an  amount  just  necessary  to  cure  the  bird  in  a  given 
time  is  found  and  then  expressing  the  vitamine  content  of  the  food 
in  terms  of  this  dosage.  In  such  an  experiment  the  value  is 
obviously  based  on  the  curative  powers  of  the  vitamine  source. 
Another  way  of  applying  the  test  is  to  determine  just  how  much 
of  the  unknown  must  be  added  to  a  diet  of  polished  rice  to  pre- 
vent the  onset  of  polyneuritic  symptoms.  Such  a  determination 
will  give  the  content  in  terms  of  preventive  dosage.  Both  methods 
have  been  extensively  applied  and  the  following  tables  compiled 
from  the  Report  of  the  British  Medical  Research  Committee 
illustrate  both  the  method  and  some  of  its  results : 

Minimum  daily  ration  thai  must  be  added  to  a  diet  of  polished  rice  to  prevent 
and  to  cure  poly-neuritis  in  a  pigeon  of  300  to  400  grams  weight.  The 
weights  given  are  in  terms  of  the  natural  foodstuff 


AMOUNT  NECESSARY 

FOR  DAILY  PREVENTION 

grams 

grams 

1.5 

Wheat  germ  (raw) 

2.5 

2.5 

Pressed  yeast 

3.0-6.0* 

30 

Egg  yolk 

eo.ot 

20.0 

Beef  muscle 

140.  Of 

3.0 

Dried  lentils 

20.  Of 

*  Autolysed.    f  Alcohol  extract. 

These  values  illustrate  both  the  method  and  its  value  in  com- 
paring sources.  Unfortunately  experience  has  shown  that  poly- 
neuritis  is  amenable  to  other  curative  agents  to  a  greater  or  less 
extent  and  it  is  difficult  to  be  sure  whether  the  curative  or  pre- 
ventive dose  represents  merely  the  vitamine  content  of  the  un- 
known or  is  the  sum  of  all  the  factors  present  in  the  curative  or 
preventive  material.  In  comparing  the  value  of  different  chem- 
ical fractions  it  probably  gives  a  fair  enough  basis  for  evaluating 
their  relative  power  but  it  is  not  entirely  satisfactory  as  a  quanti- 
tive  measure  of  vitamine  content. 

In  America  the  comparison  of  vitamine  content  has  been  largely 
based  on  feeding  experiments  with  the  white  rat.  No  other 


34  VITAMINE   MANUAL 

animal  has  been  so  well  standardized  as  this  one.  Dr.  Henry 
Donaldson  of  the  Wistar  Institute  of  Philadelphia  has  brought 
together  into  a  book  entitled  The  Eat  the  accumulated  records 
of  that  Institution  bearing  on  this  animal.  This  book  provides 
standards  for  animal  comparisons  from  every  view  point;  weight 
relation  to  age,  size  and  age,  weight  of  organs  and  age,  sex  and 
age  and  weight,  etc.  This  book  together  with  the  experience  of 
many  workers  as  they  appear  in  the  literature  and  especially  the 
observations  of  Osborne  and  Mendel  have  made  the  rat  an  ex- 
tremely reliable  animal  upon  which  to  base  comparative  data.  The 
omnivorous  appetite  of  the  animal,  his  ready  adjustment  to  con- 
finement, his  relatively  short  life  span,  all  contribute  to  his  selec- 
tion for  experimental  feeding  tests.  Another  important  reason 
for  his  selection  is  that  being  a  mammal  we  may  reasonably  con- 
sider that  his  reactions  to  foods  will  be  more  typical  of  the  human 
response  than  would  another  type,  the  bird  for  example.  It  is 
perhaps  necessary  to  sound  a  warning  here,  however,  and  point 
out  the  danger  of  too  great  faith  in  this  comparability  of  rat  and 
man  or  in  fact  of  any  animal  with  man.  In  the  case  of  the  rat 
he  has  been  found  useless  for  the  study  of  "C"  vitamine  for  the 
simple  reason  that  rats  do  not  have  scurvy.  In  general  however 
his  food  responses  to  the  vitamines,  at  least  of  the  "A"  and  "B" 
types,  have  proved,  so  far  as  they  have  been  confirmed  by  infant 
feeding,  to  be  reasonably  comparable. 

Provided  with  the  experimental  animal  the  next  step  was  to 
devise  a  basal  diet  which  should  be  complete  for  growth  in  every 
particular  except  vitamines.  Such  basal  diets  have  been  a  pro- 
cess of  development.  The  requirements  for  such  a  diet  are  the 
following  factors : 

1.  It  must  be  adequate  to  supply  the  necessary  calories  when 
eaten  in  amounts  normal  to  the  rat's  consumption. 

2.  It  must  contain  the  kinds  of  nutrients  that  go  to  make  up 
an  adequate  diet  and  in  the  per  cents  suitable  for  this  purpose. 

3.  It  must   contain  proteins  whose  quality  is  adequate  for 
growth,  i.  e.,  which  contain  the  kinds  and  amounts  of  animo  acids 
known  to  fulfil  this  function. 

4.  It  must  be  digestible  and  palatable. 


TESTING   FOE   VIT AMINES 


35 


36 


VITAMINE   MANUAL 


5.  It  must  be  capable  of  being  supplemented  by  either  or 
both  vitamines  in  response  to  the  particular  test  it  is  devised  to 
meet  and  when  both  are  present  in  proper  amounts  it  must  pro- 
duce normal  growth  and  serve  as  a  control. 


FIG.  4.    A  METABOLISM  CAGE  DEVISED  FOR  USE  IN  THE  AUTHOR'S 
LABORATORY 

The  cages  being  bottomless  are  readily  cleaned.  They  are  set  on 
circles  of  wire  mesh  over  galvanized  iron  funnels  permitting  urine  and 
feces  to  pass  through.  A  second  screen  over  the  collecting  cup  and  of 
fine  mesh  separates  the  feces  from  urine  and  also  collects  scattered  food. 

In  building  up  such  a  diet  many  experiments  have  been  com- 
bined and  thanks  largely  to  the  efforts  of  Osborne  and  Mendel 
and  McCollum  in  this  country,  we  have  a  thoroughly  standardized 
procedure  even  extending  to  types  of  cages  and  care  best  suited 
to  normal  growth  and  development.  For  clearer  appreciation 


TESTING   FOR   VITAMINES 


37 


FIG.  5.    ILLUSTRATING  THE  USE  OF  THE  CHATILLON  SCALE  FOR  RAPID 
WEIGHING  OF  ANIMALS 

The  dial  is  so  made  that  it  can  be  set  to  counterbalance  the  weight 
of  the  cage  and  the  weights  read  directly.  This  is  also  used  for  weigh- 
ing food. 


38 


VITAMINE    MANUAL 


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s 


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TESTING   FOR   VITAMINES  39 

of  the  nature  of  these  diets  and  their  preparation  we  have  sum- 
marized in  the  following  pages  the  combinations  used  by  the 
principal  contributors  to  the  subject  in  this  country. 

It  is  at  once  obvious  from  the  table  that  the  testing  value  of 
these  basal  diets  demands  the  absence  of  the  two  vitamines  iiT 
the  protein,  carbohydrates  and  fat  fractions.  To  make  sure 
of  this  absence  various  methods  have  been  devised  to  attain 
the  maximum  purity.  The  authors  recommend  the  following 
procedure: 

a.  To  purity  the  casein  or  other  protein  used.     Boil  the  pro- 
tein three  successive  times  (it  is  assumed  that  the  original  is  al- 
ready as  pure  as  it  is  possible  to  obtain  it  by  the  usual  methods  of 
preparation),  for  an  hour  each  time,  with  absolute  alcohol,  using 
a  reflux  condenser  to  prevent  loss  of  alcohol.     Filter  off  the  alco- 
hol each  time  by  suction.     This  process  will  take  off  all  the  ad- 
herent fat  and  hence  all  the  "A"  vitamine  that  might  be  present. 
The  casein  is  then  dried  and  ready  for  use.     In  certain  experi- 
ments the  authors  use  meat  residues  instead  of  a  single  protein. 
This  they  prepare  as  follows:  Fresh  lean  round  of  beef  is  run 
through  a  meat  chopper  and  then  ground  to  a  paste  in  a  Nix- 
tamal  mill,  stirred  into  twice  its  weight  of  water  and  boiled  a  few 
minutes.     The  solid  residue  is  then  strained,  using  cheese  cloth, 
pressed  in  the  hydraulic  press  and  the  cake  stirred  into  a  large 
quantity  of  boiling  water.     After  repeating  this  process  of  washing 
with  hot  water  the  extracted  residue  is  rapidly  dried  in  a  current 
of  air  at  about  60°C.     This  dried  residue  may  then  be  further 
purified  with  the  absolute  alcohol  treatment  as  described  for 
casein. 

b.  To  purify  the  carbohydrate  they  treat  starch  in  exactly  the 
same  way  as  the  casein. 

c.  To  purify  the  lard.     This  is  melted  and  poured  into  abso- 
lute alcohol  previously  heated  to  60°C.,  cooled  over  night  and 
filtered  by  suction..     This  process  is  repeated  three  times  and  the 
resulting  solids  dried  in  a  casserole  over  a  steam  bath. 

d.  When  butter  fat  is  used  to  provide  a  source  of  "A"  vitamine 
it  is  prepared  as  follows:  Butter  is  melted  in  a  flask  on  a  water 
bath  at  45°C.  and  then  centrifugated  for  an  hour  at  high  speed. 
This  results  in  a  separation  of  the  mixture  into  three  layers:  (a) 
Clear  fat  containing  the  "A"  vitamine  and  consisting  of  82  to 


40 


VITAMINE   MANUAL 


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TESTING   FOR  VITAMINES 


41 


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42  VITAMINE   MANUAL 

83  per  cent  glycerides.  This  is  siphoned  off  and  provides  the 
butter  fat  named  in  the  diets,  (b)  An  aqueous  opalescent  layer 
consisting  of  water  and  some  of  the  water-soluble  constituents 
of  the  milk.  This  is  rejected,  (c)  A  white  solid  mass  consisting 
of  cells,  bacteria,  calcium  phosphate  and  casein  particles.  This  is 
also  rejected. 

e.  When  brewers'  yeast  is  used  as  a  source  of  the  "B"  vitamine 
it  is  first  dried  over  night  in  an  oven  at  110°C.  and  then  subjected 
to  the  same  purification  process  as  the  casein  and  the  starch  to 
remove  all  trace  of  the  "A." 

The  reasons  for  the  special  precautions  just  described  have 
arisen  from  some  recent  work  of  Daniels  and  Loughlin  who  claim 
that  commercial  lard  contains  enough  "A"  vitamine  to  permit 
rats  to  grow,  reproduce  and  rear  young.  The  British  authorities 
explain  their  results  as  not  due  to  the  presence  of  the  "A"  vita- 
mine  in  the  lard  but  to  a  reserve  store  in  the  bodies  of  the  animals. 
They  hold  that  animals  may  thus  store  the  "A"  vitamine  but  that 
apparently  they  have  no  storage  powers  for  the  "B"  that  are 
comparable  to  it.  Osborne  and  Mendel  repeated  the  experiments 
described  by  Daniels  and  Loughlin,  using  the  purification  methods 
just  described,  but  failed  to  obtain  similar  results  with  either 
commercial  lard  or  with  the  purified  fraction.  They  question 
the  validity  of  the  British  explanation  but  at  the  same  time  re- 
iterate their  belief  that  even  commercial  lard  contains  no  "A" 
vitamine.  Whatever  the  explanation  of  this  particular  phenom- 
enon it  is  important  that  the  basal  diet  be  of  purified  materials 
and  the  methods  just  described  supply  the  procedure  necessary 
to  attain  that  end. 

Before  discussing  the  application  of  these  diets  to  vitamine 
testing,  attention  is  called  to  other  basal  diets  developed  by  Mc- 
Collum.  This  worker  has  paid  especial  attention  to  the  deficien- 
cies of  the  cereal  grains  and  in  particular  to  their  salt  deficiencies. 
In  his  basal  diets,  we  find,  as  would  be  expected,  special  com- 
binations particularly  suited  to  the  detection  of  vitamines  in  such 
cereals.  McCollum  has  also  devised  a  method  of  extracting  sub- 
stances to  obtain  their  "B"  vitamine  and  of  depositing  it  on  dex- 
trin. For  that  reason  he  uses  dextrin  instead  of  starch  for  his 
carbohydrate  and  when  he  wishes  to  introduce  the  "B"  vitamine 
it  can  be  done  by  his  method  without  having  to  recalculate  the 


TESTING   FOR   VITAMINES 


43 


carbohydrate  component.  His  method  consists  of  first  extracting 
the  source  with  ether  and  discarding  this  extract.  Pure  ether 
will  not  remove  the  "B"  vitamine.  The  residue  is  then  reex- 
tracted  several  times  with  alcohol  and  the  alcohol  extracts  com- 
bined. If  now  these  alcohol  extracts  are  evaporated  down  on  a 
weighed  quantity  of  dextrin  the  activated  dextrin  can  be  used  not 
only  to  supply  the  carbohydrate  of  the  ration  but  also  to  carry  the 
"B"  vitamine  of  a  given  source  that  is  under  investigation. 
McCollum's  basal  diets  and  salt  mixtures  are  tabulated  in  the 
following  chart: 

McCollum's  basal  diets  and  salt  mixtures 


INGREDIENTS 

VITAMINE  FREE 

"A"  ONLY 

"B"  ONLY 

Casein  

18.0 

57.3 
20.6 
2.0 
2.7 

18.0 
56.3 
20.0 
2.0 
3.7 

18.0 
76.3 

2.0 
3.7 

18.0 

78.3 

3.7 

18.0 
71.3 

2.0 
3.7 
5.0 

Same  as  the  vita- 
mine   free   diet 
with  "B"  add- 
ed as  yeast  as 
in  the   Mendel 
diets  or  as  ex- 
tracts    carried 
on  the  dextrin. 
In    the    latter 
case     a     given 
amount  of  dex- 
trin carries  the 
extract     of     a 
known    weight 
of  the  source  of 
the  "B" 

Dextrin       

Lactose  

Agar  .              

Salt  mixture  185  .   .   . 

Butter  fat  

Lactose  was  later  discarded  when  it  was  shown  to  be 
usually  contaminated  with  the  "B"  vitamine. 

Cereal  testing  combinations 

Wheat                    ..     .   . 

56.6 

31.5 
6.9 
5.0 

13.3 

76.4 
5.3 

5.0 

71.3 

18.0 
3.7 

5.0 
2.0 

60.0 
30.3 

4.7 
5.0 

70.0 

20.0 
5.0 
5.0 

6.0 
81.0 

6.0 
5.0 
2.0 

Wheat  embryo  

Corn  

Oats  

Skim  milk  powder  

Dextrin  

Salt  mixture  185  

Salt  mixture  314  . 

Salt  mixture  318  

Salt  mixture  500  t  .  .  . 

Salt  mixture  ?  

Butter  fat  

Agar  .  . 

44 


VITAMINE   MANUAL 


Salt  mixtures 


INGREDIENTS 

N 

UMBER  01 

MIXTURE 

3 

185 

314 

318 

500 

211 

? 

NaCl  

grams 

0.173 

grams 

1  067 

grams 

I  400 

grams 

0  5148 

grams 

0.520 

grams 

15.00 

MgSO4  anhydrous 

0  266 

1.90 

Na2HPO4:H2O 

0  347 

K2HPO4            

0.954 

3  016 

2  531 

0  3113 

34.22 

CaH4(PO4)8:H2O 

0  540 

0  276 

0.89 

Ca  lactate  

1.300 

5.553 

7.058 

2  8780 

1.971 

57.02 

Ferrous  lactate  

0  118 

K  citrate  :H2O 

0  203 

0  710 

0  5562 

0  799 

Na  citrate  anhydrous 

3  70 

Ferric  citrate  

0  100 

2.00 

Mg  citrate 

7  00 

CaCl2  

0  386 

0  2569 

CaSO4:2H20      

0  381 

0  578 

Fe  acetate  

0.100 

These  diets  fall  as  shown,  into  two  classes.  The  first  group 
correspond  to  those  of  Osborne  and  Mendel  and  are  available 
for  general  testing  of  any  unknown.  The  cereal  combinations 
are  so  constituted  that  all  deficiencies  of  salts  are  covered  and  the 
proportions  of  the  cereal  are  so  selected  as  to  provide  the  right 
proportions  of  protein,  fat  and  carbohydrate.  By  adding  enough 
butter  fat  to  supply  the  "A"  the  deficiency  in  the  "B"  can  be  tested 
and  by  adjusting  the  amounts  of  "B"  on  the  dextrin  the  cereal 
deficiency  in  this  vitamine  can  be  obtained.  It  is  obvious  that 
by  substituting  lard  for  the  butter  fat  one  could  use  the  same 
mixture  properly  supplemented  with  the  "B"  to  determine  the 
"A"  deficiencies  of  the  wheat. 

The  most  prominent  worker  in  the  field  of  the  "A"  vitamine 
measurement  in  America  is  Steenbock.  His  basal  diets  are  a 
combination  of  those  already  described. 

Steeribock's  basal  diets 

per  cent 

Casein  (washed  with  water  containing  acetic  acid) 18.0 

Dextrin 73.3 

Ether  extracted  wheat  embryo  as  source  of  vitamine  "B".    3.0 

Salt  mixture  (McCollum,  no.  185) 3.7 

Agar 2.0 


TESTING   FOR  VITAMINES 


45 


This  was  his  original  basal  diet  but  later  he  modified  it  by  adopting  the 
McCollum  method  of  carrying  his  "B"  vitamine  on  the  dextrin.  This 
was  usually  the  alcohol  extract  of  20  grams  of  wheat  embryo.  In  the  fol- 
lowing diets  the  presence  of  this  extract  is  indicated  by  the  letter  (x) 
following  the  dextrin. 


INGREDIENTS 

PER 
CENT 

18.0 

18.0 

16.0 

18.0 

16.0 

12.0 

Salt  185              

4  0 

4.0 

Salt  32    

4.0 

4.0 

2.0 

2.0 

Salt  35              

2.5 

2  5 

Dextrin  (x)                      

76  0 

71  0 

78  0 

57  0 

Butter  fat  

5  0 

5.0 

Beets  

15.0 

Potatoes               

79  5 

83.5 

Agar                

2  0 

2.0 

2  0 

1  0 

Steenbock's  salt  mixtures 

McCollum's  no.  185;  see  page  44. 

No.  32  consisted  of:  grams 

NaCl 0.202 

Anhydrous  MgSO4 0.311 

K2HPO4 1.115 

Ca  lactate 0.289 

Na2HPO4:12H2O 0.526 

Ca2H2(PO4)2:H2O 1.116 

Fe  citrate 0.138 

No.  35  consisted  of: 

NaCl 1 .00 

CaCO3 1.5 

The  very  nature  of  these  basal  diets  suggests  their  use.  In 
general  however  their  utilization  for  testing  purposes  is  based  on 
the  following  principles:  Since  the  basal  diet  supplies  all  the  re- 
quirements of  a  food  except  the  vitamine  for  which  one  is  testing, 
it  is  simply  necessary  to  add  the  unknown  substance  as  a  given  per 
cent  of  the  diet  and  observe  the  results.  If  the  amount  added  is 
small  it  is  assumed  that  its  addition  will  not  appreciably  effect  the 
optimum  concentrations  of  nutrients,  etc.,  and  for  such  experi- 
ments no  allowances  are  made  for  the  constituents  in  the  un- 


46  VITAMINE   MANUAL 

known.  For  example  let  us  assume  that  we  wish  to  test  the  value 
of  a  yeast  cake  as  a  source  of  "B"  vitamine.  We  first  select  a 
sufficient  member  of  rats  of  about  thirty  days  age  to  insure  pro- 
tection from  individual  variations  in  the  animals.  The  age  given 
is  taken  as  an  age  when  the  rats  have  been  weaned  and  are  capable 
of  development  away  from  the  mother  and  as  furnishing  the  period 
of  most  active  growth.  These  rats  are  now  placed  on  one  of  the 
basal  diets  which  in  this  case  supplies  all  the  requirements  except 
the  "B"  vitamine.  In  this  experiment  any  of  the  diets  of  Osborne 
and  Mendel  or  of  McCollum  will  do  that  have  been  labelled  "A" 
only.  After  a  week  or  so  on  this  diet  they  will  have  cleared  the 
system  of  the  influence  of  previous  diets  and  their  weight  curves 
will  be  either  horizontal  or  declining.  If  now  we  make  the  diet 
consist  of  this  basal  diet  plus  say  5  per  cent  of  yeast  cake,  the 
weight  curve  for  the  next  few  weeks  will  show  whether  that 
amount  supplies  enough  for  normal  growth,  comparison  being 
made  with  the  normal  weight  curve  for  a  rat  of  that  age. 

In  this  method  it  is  assumed  that  the  amount  of  yeast  cake 
added  will  not  derange  the  proportions  of  protein  fat,  etc.,  in  the 
basal  diet  enough  to  affect  optimum  conditions  in  these  respects. 
This  is  a  curative  type  of  experiment.  If  we  wish  to  develop  a 
preventive  experiment  the  yeast  cake  may  be  incorporated  in  the 
diet  from  the  first  and  the  amount  necessary  to  prevent  deviation 
from  the  normal  curve  determined.  Both  methods  are  utilized, 
the  one  checking  the  other.  If  however  the  amount  of  the  sub- 
stance necessary  to  supply  the  vitamine  required  for  normal 
development  is  large  such  addition  would  of  course  disturb  the 
proportions  of  nutrients  in  the  normal  diet  and  in  that  case  anal- 
ysis must  be  made  of  the  substance  tested  to  determine  its 
protein,  fat,  carbohydrate  and  salt  content  and  the  basal  diet 
corrected  from  this  viewpoint  so  as  to  retain  the  optimum  propor- 
tions of  these  factors.  McCollum's  cereal  testing  combinations 
are  illustrative  of  such  methods  applied  to  cereals.  Still  another 
method  is  to  add  a  small  per  cent,  of  the  unknown  and  then  add 
just  enough  of  the  vitamine  tested  to  make  sure  that  normal 
growth  results.  Such  a  method  gives  the  results  in  terms  of  a 
known  vitamine  carrier.  For  example,  if  we  add  to  a  basal  diet, 
sufficient  in  all  but  the  "A"  vitamine  (Steenbock's  mixture  for 


TESTING   FOB  VITAMINES  47 

example),  a  small  per  cent  of  a  substance  whose  content  in  "A" 
is  unknown  and  note  that  growth  fails  to  result  we  can  then  add 
butter  fat  until  the  amount  just  produces  normal  growth.  If 
now  we  know  just  what  amount  of  butter  fat  suffices  for  this  pur- 
pose when  used  alone  we  can  calculate  the  part  of  the  butter 
which  is  replaced  by  the  per  cent  of  unknown  used.  To  put  this 
in  terms  of  figures  will  perhaps  make  the  idea  clearer.  Let  us 
assume  that  5  per  cent  of  butter  fat  in  a  given  diet  is  sufficient 
to  supply  the  "A"  necessary  for  normal  growth.  Assume  that 
the  addition  of  5  grams  of  the  unknown  in  100  grams  of  the  butter- 
free  diet  fails  to  produce  normal  growth  but  that  by  adding  2  per 
cent  of  butter  fat  normal  growth  is  reached.  It  is  obvious  under 
these  conditions  that  5  grams  of  the  unknown  is  equivalent  in 
"A"  vitamine  content  to  5  minus  2  grams  of  butter  fat,  i.e.,  is 
equivalent  to  3  grams  of  butter  fat  or  expressed  in  per  cents  the 
substance  contains  0.6  or  60  per  cent  of  the  "A"  found  in  pure 
butter  fat. 

Experience  has  shown  that  it  is  dangerous  to  draw  conclu- 
sions from  experiments  of  too  short  duration  or  to  base  them  on 
too  few  animals.  For  complete  data  the  experiments  should  be 
carried  through  the  complete  life  cycle  of  the  rat,  including  the 
reproductive  period.  Otherwise  it  may  turn  out  that  the  amount 
in  the  unknown  while  apparently  sufficient  for  normal  growths  is 
incapable  of  sustaining  the  drain  made  in  reproduction.  It  is 
this  consideration  that  makes  the  accumulation  of  authoritative 
data  on  vitamine  contents  of  foodstuffs  so  slow  and  tedious  and 
one  of  the  reasons  why  we  lack  satisfactory  tables  in  this  partic- 
ular at  present.  Osborne  and  Mendel  raise  another  point  of 
methodology  and  believe  that  more  accurate  results  will  be  ob- 
tained if  the  source  of  the  vitamine  is  fed  separately  than  if  mixed 
with  the  basal  diet .  It  is  easily  possible  that  since  one  of  the  effects 
of  lack  of  vitamine,  especially  of  the  "B"  type,  is  poor  appetite, 
the  amount  necessary  to  produce  normal  growth  may  be  smaller 
than  would  appear  from  results  obtained  by  mixing  it  in  the  basal 
diet.  When  so  mixed  the  animals  do  not  get  enough  to  maintain 
appetite  and  really  decline  because  they  do  not  eat  enough  rather 
than  because  the  amount  of  vitamine  given  is  inadequate  to  growth. 
Details  of  this  kind  are  matters  however  that  particularly  concern 


48  VITAMINE   MANUAL 

the  experimentalist  and  as  our  purpose  here  is  to  merely  describe 
the  methodology  we  may  perhaps  turn  now  to  other  types  of  test- 
ing. Before  doing  so  it  is  perhaps  unnecessary  to  suggest  that  in 
all  experiments  it  is  important  that  the  food  intake  consumed  be 
measured.  Also  that  in  all  such  experimentation  it  is  necessary 
to  run  controls  on  a  complete  diet  rather  than  to  rely  too  much 
on  standard  figures.  For  this  latter  purpose  it  is  merely  neces- 
sary to  add  to  the  basal  diets  the  "A"  as  butter  fat  and  the  "B" 
as  dried  yeast  or  otherwise  to  make  them  complete.  Various 
special  mixtures  have  been  tested  out  for  this  purpose  and  the 
data  already  presented  supplies  the  information  necessary  to 
construct  such  control  diets.  Professor  Sherman  has  given  me 
the  following  as  a  control  diet  on  which  he  has  raised  rats  at  normal 
growth  rate  to  the  fifth  generation: 

One-third  by  weight  of  whole  milk  powder. 
Two-thirds  by  weight  of  ground  whole  wheat. 

Add  to  the  mixture  an  amount  of  NaCl  equal  to  2  per  cent  of  the  weight 
of  the  wheat. 

A  control  mixture  based  on  Osborne  and  Mendel's  data  would 
have  the  following  components: 


Meat  residue  19.6  per  cent  or  casein  18  per  cent. 

Starch  52.4  per  cent  or  49  per  cent. 

Lard  15  per  cent  or  20  per  cent. 

Artificial  protein-free  milk  4  per  cent. 

Butter  fat  9  per  cent. 

Dried  yeast  0.2  to  0.6  gram,  daily. 


The  preceding  description  has  applied  especially  to  testing  for 
the  presence  of  the  "A"  or  the  "B"  vitamine.  When  we  come 
to  the  methods  of  testing  for  the  "C"  type  it  is  necssary  to  change 
our  animal.  Rats  do  not  have  scurvy  but  guinea  pigs  do.  The 
philosophy  of  the  tests  for  the  antiscorbutic  vitamines  then  will 
be  identical  with  that  of  the  polyneuritic  methods  with  pigeons,  viz., 
preventive  and  curative  tests  with  guinea  pigs.  The  "C"  vita- 
mine  is  especially  sensitive  to  heat  and  this  fact  enables  us  to 
secure  a  "C"  vitamine-free  diet.  LaMer,  Campbell  and  Sherman 
describe  their  methods  as  follows: 


TESTING   FOR   VITAMINES  49 

First  select  guinea  pigs  of  about  300  to  350  grams  weight. 
Test  these  with  the  basal  diet  until  you  secure  pigs  that  will  eat 
the  diet.  Those  that  will  not  eat  it  at  first  are  of  no  use  for 
testing  purposes,  for  a  guinea  pig  will  starve  to  death  rather  than 
eat  food  he  doesn't  like.  Having  secured  pigs  that  will  eat  they 
should  on  a  suitable  basal  diet  die  of  acute  scurvy  in  about  twenty- 
eight  days.  Their  basal  diet  is  as  follows: 

Skim  milk  powder  heated  for  two  hours  at  110°C.  in  an  air  percent 
bath  to  destroy  the  "C"  vitamine  that  might  be  present ...   30 

Butter  fat 10 

Ground  whole  oats 59 

NaCl 1 

They  claim  that  when  fruit  juice  addenda  are  given  in  minimal 
protective  doses  and  calculated  to  unit  weight  bases,  the  results 
are  comparable  in  precision  to  those  of  antitoxin  experiments. 

Old  food  should  be  removed  every  two  days  and  replaced  by 
new,  cups  being  cleaned  at  the  same  time.  Since  this  is  a  scurvy- 
producing  diet  its  use  is  obvious.  We  can  let  the  pig  develop 
scurvy  on  it  and  then  test  the  curative  powers  of  the  unknown  by 
adding  it  to  the  diet  or  we  can  add  it  to  the  diet  from  the  first 
and  determine  the  dose  necessary  to  prevent  scurvy;  or  we  can 
determine  its  effect  in  terms  of  a  known  antiscorbutic  such  as 
orange  juice  by  combining  it  with  measured  quantities  of  the 
orange  juice. 

There  are  other  diets  that  have  been  given  for  this  purpose, 
e.g.,  Hoist  and  Frohlich  induced  scurvy  by  restricting  animals  to 
an  exclusive  diet  of  cereals  (oats  or  rye  or  barley  or  corn).  Hess 
and  linger  have  used  hay,  oats  and  water  given  ad  libitum.  All 
of  these  and  others  are  subject  to  criticism  on  the  basis  that  they 
are  not  necessarily  adequate  in  other  food  factors  and  may 
therefore  not  be  fair  bases  for  testing  the  antiscorbutic  powers 
of  the  unknown  combined  with  them.  Abels  has  recently  shown 
that  scurvy  increases  susceptibility  to  infections  and  believes 
that  the  scurvy  hemorrhages  are  brought  about  by  the  toxic 
effects  of  infection.  It  is  therefore  desirable  in  testing  for  anti- 
scorbutic power  that  the  basal  diet  be  itself  as  complete  as  pos- 
sible in  all  factors  except  the  absence  of  "C." 


50  VITAMINE   MANUAL 

The  study  of  rickets  has  already  progressed  to  the  stage  of  cal- 
culating rickets-producing  diets  and  the  methodology  is  identical 
with  that  for  scurvy  but  this  phase  of  testing  still  lacks  evidence 
of  an  antirachitic  vitamine  and  in  that  uncertainty  it  is  hardly 
worth  while  to  elaborate  these  diets  here.  The  British  diets  are 
all  based  on  Mellanby's  contention  that  the  "A"  vitamine  is  the 
antirachitic  vitamine.  This  view  is  not  yet  accepted  by  Amer- 
ican workers. 

In  concluding  this  chapter  it  is  sufficient  to  state  that  with  our 
present  methodology  the  accumulation  of  data  for  evaluating  the 
vitamine  content  of  various  foods  is  still  far  from  satisfactory 
and  from  the  chemist's  viewpoint  the  methodology  is  most  unsatis- 
factory as  a  means  of  testing  fractional  analyses  obtained  in  the 
search  for  the  nature  of  the  substance,  both  because  of  the  time 
consumed  in  a  single  test  and  from  the  difficulty  of  using  the 
fractions  in  feeding  experiments  when  these  fractions  may  them- 
selves be  poisonous  or  otherwise  unsuited  for  mixture  in  a  diet. 
It  is  obvious  therefore  that  interest  is  keen  in  any  possibility  of 
devising  a  test  that  will  be  specific,  quick  and  not  require  modi- 
fication of  the  material  tested,  because  of  its  unsuitability  for 
feeding.  In  1919  Roger  J.  Williams  proposed  a  method  that 
seemed  to  offer  promise  in  these  respects  but  which  is  not  yet 
in  the  form  for  quantitative  use.  It  offers  promise  that  entitles 
it  to  a  special  chapter  for  discussion  and  the  next  chapter  presents 
the  present  status  of  the  so-called  yeast  test  for  vitamine  "B." 

Before  turning  to  this  test  it  is  well  to  call  attention  here  to 
the  importance  of  the  experimental  animal.  Without  the  poly- 
neuritic  fowls  we  might  never  have  cured  beri-beri,  the  guinea 
pig  made  the  solution  of  the  scurvy  problem  possible  and  if  some 
way  of  inducing  pellagra  in  an  animal  can  be  devised  that  scourge 
may  yet  be  eliminated. 


CHAPTER  IV 
THE  YEAST  TEST  FOR  VITAMINE  "B" 

As  far  back  as  the  days  of  Pasteur  a  controversy  arose  over 
the  power  of  yeast  cells  to  grow  on  a  synthetic  medium  composed 
solely  of  known  constituents.  This  controversy  hinged  on  a  dis- 
cussion as  to  whether  these  media  were  efficient  unless  reinforced 
with  something  derived  from  a  living  organism.  In  1901  Wildier 
in  France  published  an  article  in  which  he  showed  that  extracts 
of  organic  matter  when  added  to  synthetic  media  had  the  power 
to  markedly  stimulate  the  growth  of  yeast  organisms.  He  did 
not  attempt  at  the  time  to  identify  the  nature  of  this  stimulatory 
substance,  but  since  it  was  derived  from  living  organisms,  he 
called  it  "Bios."  Soon  after  the  discovery  of  vitaminesthe 
bacteriologists  began  to  discover  that  they  or  an  analgous  factor 
apparently  played  a  part  in  the  growth  of  certain  strains  of  bac- 
teria, especially  the  meningococcus.  In  1919  Roger  Williams 
working  in  Chicago  University  was  struck  with  the  bearing  of 
Wildier's  work  on  the  vitamine  hypothesis  and  formed  the  theory 
that  Wildier's  "bios"  might  be  the  water-soluble  vitamine  "B." 
He  proceeded  to  test  out  this  theory  and  demonstrated  that 
extracts  of  substances  rich  in  the  "B"  vitamine  had  a  marked 
effect  on  the  stimulation  of  yeast  growth.  He  developed  these 
experiments  and  devised  a  method  of  comparing  the  growth  of 
yeast  cells  when  stimulated  by  such  extracts.  The  results  were 
so  striking  as  to  appear  to  justify  his  view  and  he  then  suggested 
that  his  method  might  be  used  as  a  test  for  the  measure  of  "B" 
vitamine  in  a  given  source.  William '&  method  consisted  essen- 
tially in  adding  the  extract  of  an  unknown  substance  to  hanging 
drops  in  which  were  suspended  single  yeast  cells  and  observing 
the  rate  of  growth  under  the  microscope.  Soon  after,  Miss 
Freda  Bachman  reinvestigated  the  problem  with  various  types 
of  yeast  and  found  that  practically  all  types  of  yeast  respond  to 
the  stimulation  of  these  "bios"  extracts.  Her  method  consisted 
in  the  use  of  fermentation  tubes  and  the  stimulatory  effect  was 

51 


52  VITAMINE   MANUAL 

measured  by  the  amount  of  C02  produced  in  a  given  time.  By 
this  method  she  confirmed  Williams'  view  that  the  "bios"  of 
Wildier  was  apparently  identical  with  vitamine  "B"  and  that 
most  yeasts  require  this  vitamine  for  their  growth.  She  also 
suggested  that  her  method  might  be  made  the  basis  of  a  test  for 
vitamine  content.  In  1919  Eddy  and  Stevenson  made  extended 
experiments  with  these  two  methods  in  the  attempt  to  improve 
the  technique  and  make  it  serve  as  a  quantitative  measure. 
Their  experiments  served  two  purposes,  first  to  bring  out  certain 
difficulties  in  the  methods  of  the  two  authors  from  the  quantita- 
tive viewpoint  and  the  development  of  a  technique  to  correct 
these  difficulties  and  secondly  to  add  more  data  bearing  on  the 
specificity  of  the  test.  Soon  after  their  publication  Funk  became 
interested  and  coming  to  the  same  conclusions  as  to  specificity 
devised  a  centrifugating  method  for  measuring  the  yeast  growth. 
Williams  also  improved  his  original  method  and  devised  a  gravi- 
metric method  for  the  same  purpose.  From  the  viewpoint  of 
methodology  we  now  have  methods  which  are  suitable  as  quanti- 
tive  procedures  for  determining  the  effect  of  extracts  of  unknown 
substances  on  yeast  growth  and  hence  if  the  stimulatory  sub- 
stance is  vitamine  "B,"  a  means  of  determining  within  a  space 
of  twenty-four  hours  the  approximate  content  of  stimulatory 
material  in  a  given  source.  Since  the  Funk  method  is  the  sim- 
plest of  these  and  illustrates  the  principles  involved  it  will  suffice 
to  describe  that. 

Funk  method  of  yeast  test  vrith  Eddy  and  Stevenson  modification 

1.  To  a  basal  diet  of  9  cc.  of  sterile  culture  medium  such  as  a  von  Nageli 
solution1  in  a  sterile  test  tube  is  added  1  cc.  of  the  sterile,  neutral,  watery 
extract  of  the  source  of  the  vitamine.  A  pure  culture  of  Fleischman's 
yeast  (Funk  prefers  brewer's  yeast)  is  maintained  on  an  agar  slant  and 
twenty-four  hours  before  the  test  is  to  be  made,  a  transplant  is  made  to  a 
fresh  agar  slant.  One  standardized  platinum  loopful  of  the  twenty-four 

1  von  Nageli's  solution  consists  of  the  following  ingredients  NH4NO3, 
1  gram;  Ca3(PO4)2,  0.005  gram;  MgSO4,  0.25  gram  dextrose.  10.0  grams 
made  up  to  100  cc.  with  distilled  water.  Other  culture  media  may  be  used 
and  such  combinations  will  be  found  in  any  text  on  yeasts.  They  all 
permit  a  certain  amount  of  growth  but  all  are  apparently  stimulated  by 
the  addition  of  vitamine  extracts. 


YEAST  TEST   FOR   VITAMINE    "fi"  53 

hour  yeast  growth  is  then  used  to  inoculate  the  contents  of  the  tube,  the 
tube  stoppered  with  cotton  and  incubated  for  from  twenty-four  to  seventy- 
two  hours  at  a  temperature  of  31  °C.  The  seventy-two  hour  incubation 
period  yields  nearly  optimum  growth  for  this  purpose. 

2.  At  the  end  of  this  time  the  yeasts  are  killed  by  plunging  the  tube  in 
water  heated  to  80°C.  and  maintained  at  this  temperature  for  fifteen 
minutes.  The  contents  of  the  tubes  are  then  poured  into  a  Hopkins  cen- 
trifuge tube  which  has  a  capillary  tip  graduated  in  hundredths  of  a  cubic 
centimeter.  After  twenty  minutes  centrifugating  at  a  speed  of  about  2400 
revolutions  per  minute  the  yeasts  in  the  solution  have  all  been  packed 
into  the  tip  and  the  volume  can  then  be  read  accurately  to  thousandths 
of  a  cubic  centimeter  (with  the  aid  of  a  scale  and  magnifier).  With  a 
control  tube  containing  9  cc.  of  the  sterile  media  and  1  cc.  of  distilled 
water  in  place  of  the  1  cc.  of  extract  a  comparison  can  be  obtained  which 
is  an  accurate  measure  of  the  stimulatory  effect  of  the  extract.  If  this 
stimulus  is  due  purely  to  vitamine  it  is  obvious  that  this  procedure  would 
enable  us  to  compare  extracts  of  known  weights  of  and  arrive  at  compari- 
sons which  would  be  measures  of  their  vitamine  content.  In  other  words 
the  procedure  is  now  in  a  satisfactory  form  for  testing  and  its  value  depends 
merely  upon  our  ability  to  show  that  the  stimulus  given  the  yeast  is  due 
solely  to  vitamine  "B." 

The  interest  of  the  vitamine  student  in  this  test  will  be  easily 
understood  for  it  is  so  simple  of  manipulation  and  so  rapid  in 
producing  results  that  it  is  the  nearest  approach  to  a  chemical 
test  of  satisfactory  nature  yet  proposed  but  unfortunately  evi- 
dence soon  began  to  accumulate  to  show  that  the  stimulation 
produced  by  extracts  of  various  sources  is  not  a  matter  of  pure 
vitamine.  If  we  plot  a  curve  of  stimulation  for  various  dilutions 
of  a  given  extract  we  find  that  the  stimulation  is  not  directly 
proportional  to  the  concentration  of  vitamine  present  but  is  a 
composite  of  several  factors.  The  chart  derived  from  experi- 
ments by  Eddy  and  Stevenson  shows  the  general  nature  of  this 
curve.  Other  experimenters  have  reached  similar  results  and 
some  have  gone  so  far  as  to  maintain  that  the  stimulation  is  not 
due  to  vitamine  "B"  at  all.  It  is  therefore  evident  that  until 
this  controversy  is  settled  the  yeast  test  cannot  be  used  for  the 
purpose  proposed.  Our  own  experiments  at  present  make  us 
still  firm  in  our  belief  that  one  of  the  factors  and  perhaps  the  most 
important  factor  in  the  stimulation  effect  is  the  vitamine  but 
until  we  can  devise  a  basal  medium  that  is  comparable  to  that 
used  in  rat  feeding  experiments,  i.e.,  one  that  contains  all  the 


54 


VITAMINE   MANUAL 


elements  for  optimum  growth  of  yeasts  except  vitamine  "B"  it 
will  be  unsafe  to  draw  conclusions  from  the  test  as  to  vitamine 
content.  It  may  be  possible  to  so  treat  our  extracts  as  to  elim- 
inate from  them  all  other  stimuli  except  the  vitamine  or  to 


VflRllNlj-COMtLNTRRTIDNS  Of-flLrftLFil-QTRillT 


.03 


X 


0.1 


0.1 


0.3 


0.1 


If 


U> 


0.1     MT 


CONCE»TRHTI(IN5TDTtl> 


FIG.  7.  GROWTH  RATE  OF  YEAST  UNDER  ALFALFA  EXTRACT  STIMULATION 

This  chart  shows  the  effect  of  varying  concentrations  of  an  alfalfa 
extract  on  the  growth  rate  of  the  yeast  cell.  The  rate  of  growth  was 
determined  after  the  Funk  method  by  centrifuging  the  cells  after  seventy- 
two  hours  incubation  and  measuring  the  volume  in  cubic  centimeters. 
The  shape  of  the  curve  shows  that  this  method  will  not  give  comparative 
results  unless  the  extracts  tested  are  dilute  enough  for  the  determinations 
to  fall  in  the  steep  part  of  the  curve. 

destroy  the  vitamine  in  them  and  thus  permit  the  comparison  of 
an  extract  with  the  vitamine  destroyed  against  one  in  which  it 
is  present  and  thus  arrive  at  the  result  desired.  At  any  rate  all 
we  can  say  at  present  is  that  the  yeast  test  is  unreliable  as  a 


YEAST  TEST  FOR  VITAMINE   "B"  55 

measure  of  vitamine  content  but  that  if  it  can  be  made  quantita- 
tive its  advantages  are  so  great  that  it  is  very  much  worth  while 
to  continue  work  upon  it  until  it  is  certain  that  it  cannot  be  made 
to  produce  the  desired  result. 

Another  reason  for  our  attention  to  this  test  is  that  if  it  can  be 
made  to  show  vitamine  effect  it  provides  an  excellent  medium  for 
investigation  of  vitamine  "B"  reactions,  and  a  method  for  study- 
ing the  effect  of  the  vitamine  upon  the  protoplasm  of  a  single 
cell. 


CHAPTER  V 


THE  SOURCES  OP  THE  VITAMINE 

Having  now  considered  the  general  principles  involved  in  vita- 
mine  testing  we  may  justly  ask  what  information  they  have 
yielded  us  in  regard  to  the  distribution  of  the  vitamines  in 
nature.  If  we  must  include  vitamines  in  our  diets  it  is  important 
to  know  how  to  select  foods  on  this  basis,  hence  a  classification  of 
them  on  the  ground  of  vitamine  distribution  becomes  essential. 
The  newness  of  the  subject  and  the  limited  tests  that  have  been 
made  as  well  as  the  uncertainty  residing  in  the  test  results  make 
any  classifications  presented  more  or  less  approximations  but  we 
present  such  attempts  as  have  been  made,  with  the  understanding 
that  these  tabulations  are  merely  guides  and  not  quantitative 
measurements  in  the  sense  that  tables  giving  calorie  values  of 
protein,  fat  and  carbohydrate  content  are.  The  following  table  (1) 
has  been  freely  copied  from  a  report  of  the  British  Medical 
Research  Committee  to  which  acknowledgment  is  hereby  given. 

TABLE  i 
Pages  50  and  51  of  the  British  Medical  Research  Committee's  report 


CLASSES  OF  FOODSTUFFS 

VITAMINE  "A" 

VITAMINE  "B" 

VITAMINE  "C" 

Fats  and  oils: 
Butter  

+  +  + 

o 

Cream  

+  + 

0 

Cod-liver  oil        

+  +  + 

o 

Mutton  and  beef  fat  or  suet.  .  . 
Lard  

+  + 

0 

Olive  oil            

o 

Cotton  seed  oil  

0 

0 

Cocoa-butter       

0 

Linseed  oil  

0 

The  following  table  (2)  has  been  compiled  from  a  review  of  both 
British  and  American  data  and  represents  a  rather  more  complete 
classification  than  the  British  report.  The  four  plus  system  has 
also  been  used  to  permit  more  complete  comparisons. 

56 


TABLE  1— Continued 


CLASSES  OF  FOODSTUFFS 

VITAMINB  "A" 

VITAMINB  "B" 

VITAMINB  "c" 

Fats  and  oils  —  continued: 
Fish  oil,  whale  oil,  herring  oil, 
etc                

+4- 

Hardened  fats  (hydrogenated) 
of  animal  or  vegetable  origin 
Margarine  from  animal  fat  .... 

Margarine  from  vegetable  fat 
or  lard  

0 

In  propor- 
tion to 
animal 
fat  used 

0 

Nut  butters  

+ 

Meat,  fish,  etc.: 
Lean  meat  (beef,  mutton,  etc.) 
Liver  

4- 
4-+ 

4- 
4-4- 

•f 

4- 

Kidneys  

4-4- 

4- 

Heart  

4-4- 

4- 

Brain  

4- 

4-4- 

Sweetbreads     

4- 

4-4- 

Fish,  white  

0 

Very  slight 

• 

Fish  fat  (salmon,  herring,  etc.) 
Fish  roe  

4~h 
+ 

if  any 
Very  slight 
if  any 
4-4- 

Tinned  meats  

? 

Very  slight 

0 

Milk,  cheese,  etc.: 
Milk,  cow's  whole  raw 

+4- 

+ 

+ 

Milk,  cow's  skim  

0 

+ 

+ 

Milk,  cow's  dried  whole 

Less  than 

4. 

Less  than 

Milk,  cow's  boiled  whole  

4-4- 
? 

4- 

4- 
Less  than 

Milk,  cow's  condensed  sweet- 
ened .          .  .        

4. 

4- 

4- 

Cheese,  whole  milk  

4. 

Less  than 

Cheese,  skim  milk  

o 

4- 

Eggs,  fresh  

4-4- 

4-4-4- 

0? 

Eggs,  dried  

4-4- 

4-4-4- 

0? 

Cereals,  pulses,  etc.: 
Wheat,    maize,    rice    (whole 
germ) 

4- 

4- 

o 

Wheat,  maize,  rice  germ  

-f  4- 

4-4-4- 

0 

Wheat,  maize,  rice  bran  .... 

0 

4-4- 

o 

White  wheat  flour,  pure  corn 
flour,  polished  rice,  etc  

0 

0 

0 

57 


TABLE  1— Continued 


CLASSES  OF  FOODSTUFFS 

VITAMINE  "A" 

VITAMINE  "fl" 

VITAMINB  "c" 

Cereals,  pulses,  etc.  —  continued: 
Custard  powders,  egg  substi- 
tutes, prepared  from  cereal 
products 

0 

o 

o 

Linseed,  millet      

4-4- 

4-4- 

0 

Dried  peas  lentils   etc 

4-4- 

Pea-flour,  kilned  

0 

0 

Soy  beans  haricot  beans  

+ 

4-4- 

o 

Germinated  pulses  or  cereals  .  . 
Vegetables  and  fruits: 
Cabbage  fresh,  raw        

4- 
+-f 

4-4- 
-f 

++ 

4-  -1-4- 

C^ibba^e  fresh  cooked 

-j- 

4- 

Cabbage,  dried        

+ 

-f- 

Very  slight 

Cabbage   canned           .       .   . 

Very  slight 

Swedes,  raw  expressed  juice  .  .  . 
Lettuce                    

4-4- 

+ 

+++ 

Spinach,  dried  

4-4- 

4- 

Carrots  fresh,  raw    . 

-f 

4- 

+ 

Carrots   dried 

Very  slight 

Less  than 

Beetroot,  raw,  expressed  juice 
Potatoes,  raw  

+ 

+ 

+ 

+ 

Potatoes,  cooked     

+4- 

Beans,   fresh  scarlet  runners 
raw          

Lemon  juice  fresh                ,  .  . 

++-h 

Lemon  juice,  preserved  

Lime  juice  fresh           

++ 

Lime  juice  preserved 

Very  slight 

Orange  juice,  fresh    

+++ 

Raspberries 

+  + 

Apples    . 

+ 

Bananas                       

-|~ 

4. 

Very  slight 

++ 

Nuts      .  .          

4- 

4-4- 

Miscellaneous: 
Yeast  dried  ,  

? 

4-4-4- 

Yeast  extract  and  autolysed.  .  . 
Meat  extract                  .   . 

? 
0 

+++• 
0 

0 
0 

Malt  extract  

•+•  in  some 

Beer 

specimens 
0 

0 

Honey  

-f 

4-4-4-  indicates  abundant;  4- 4-  relatively  large;  4-  present  in  small 
amount;  0  absent. 

58 


SOURCES   OF  VITAMINES 


59 


TABLE  2 


FOODSTTJOT 

"A" 

*'B" 

"c" 

Meats: 
Beef  heart  

4. 

+ 

j 

Brains  

4-4- 

4.4-4. 

+? 

Codfish 

4- 

+ 

? 

Codtestes  

+ 

Fish  roe  

+ 

4-4- 

? 

Herring  

4-4- 

4-4- 

? 

Horse  meat  

+ 

4- 

Kidney  

4-+ 

4-4- 

Lean  muscle  

0 

o 

4-? 

Liver  

+ 

-4- 

4-? 

Pancreas  

o 

4-4-4- 

Pig  heart  

-f 

4- 

7 

Placenta  ,  

+ 

Thymus  (sweetbreads) 

o 

o 

o 

Vegetables: 
Beet  root  

4- 

4- 

++ 

Beet  root  juice  

? 

Little 

4-4-+ 

Cabbage,  dried  

++4- 

4-4-4- 

4_ 

Cabbage,  fresh  

4-4-4- 

4-4-4. 

++++ 

Carrots  

+  4-4- 

4-4-4- 

4  —  1- 

Cauliflower  

4-4- 

4-4-4- 

++ 

Celery  

? 

4-4-+ 

J 

Chard  

4-4-4- 

+4- 

f 

Dasheens  

+ 

4-+ 

? 

Lettuce  

4.4- 

4-4- 

++++ 

Mangels  

4-4- 

4-4- 

? 

Onions  

? 

4-4-4- 

+++ 

Parsnips  

4-4- 

++4- 

Peas  (fresh)  

4- 

4-4- 

4-++ 

Potatoes  

o 

+  4-+ 

4-4- 

Potatoes  (sweet)  

4-4-4- 

4-4- 

? 

Rutabaga  

+  4-+ 

Spinach  

+++ 

4-++ 

++4- 

Cereals: 
Barley  

4- 

4-4-4- 

7 

Bread  (white)  

4-? 

Bread  (whole  meal)  

-u 

4-4-4- 

7 

Maize  .  .                                       < 

-f-  In  yellow 

>     4-4-4- 

7 

1 
Oats  

0  In  white 

4- 

4-4-4- 

o 

Rice  polished  

0 

o 

o 

Rice  (whole  grain)  

4- 

+  +  4- 

o 

Rye  

4- 

4-4-4- 

o 

60 


VITAM1NE   MANUAL 


TABLE  2—  Continued 


FOODSTUFF 


Cereals — continued : 

Corn  embryo 

Corn  (kaffir) 

Corn  (see  maize) 

Corn  pollen 

Malt  extract 0 

Wheat  bran 0 

Wheat  embryo 

Wheat  endosperm 0 

Wheat  kernel + 

Other  seeds: 

Beans,  kidney 

Beans,  navy 

Beans,  soy + 

Cotton  seed 

Flaxseed -f-|- 

Hemp  seed +4- 

Millet  seed 

Peanuts 

Peas  (dry) +? 

Sun  flower  seeds 
Fruits: 

Apples 

Bananas ? 

Grapefruit 

Grape  juice 

Grapes 0 

Lemons 

Limes 

Oranges 

Pears 

Raisins 

Tomatoes +-f 

Oils  and  fats: 

Almond  oil 0 

Beef  fat + 

Butter ++++ 

Cocoanut  oil 0 

Cod  liver  oil 

Corn  oil 0 

Cotton  seed  oil 0? 

Egg  yolk  fat ++++ 

Fish  oils 

Lard..  0? 


H-+ 

+ 
+ 
+ 


SOURCES  OP  VITAMINES 


61 


TABLE  2— Continued 


FOODSTUFF 

"A" 

•V 

"c" 

Oils  and  fats  —  continued: 
Oleo,  animal  

+ 
0 
0 
0? 
0 
0? 

+ 
++ 

+ 
+ 

++++ 
++ 

4-+ 
+-H- 
++++ 
+ 
+++ 
+++ 
+ 

+++ 
Va 
+++ 

0 
0 
++ 
0 
0 
0 

0 
0 
0 
0 
0 
0 

+++ 
+4H- 
+++ 

H~H- 
+-f+ 
++-h 
+ 

+ 

0 

+ 
-f 

+ 

+4- 
+++ 
+++• 
+++ 
+++ 

+++ 
ries  with  sou 
++++ 
++ 
0 
0 
+++ 
++++ 
++ 
+++ 

0 
0 
0 

0 
0 

+ 
+ 

0 

? 

0 

? 

0 

+? 
-f? 
++ 
+ 

? 

rce 
? 
0 
0 
0 

0 
0 
0 

Olive  oil  

Pork  fat  

Tallow  

Nuts: 

Brazil  nut  

Cocoanut   .    .  .       

English  walnuts  

Filbert  

Hickory                             

Pine  

Dairy  products: 
Butter  

Cheese    

Condensed  milk  

Eges  ,  . 

Milk  powder  (whole)  

Milk  whole    

Whey.. 

Miscellaneous: 
Alfalfa  ,  

Blood  

Clover  

Malt  extract          

Nectar  

Timothy    

Yeast  cakes         

CHAPTER  VI 
THE  CHEMICAL  AND  PHYSIOLOGICAL  PROPERTIES  OF  THE  VITAMINE 

While  the  chemists  have  not  yet  been  able  to  isolate  and  iden- 
tify the  various  vitamines  they  have  succeeded  in  demonstrating 
many  of  the  properties  of  these  substances  and  it  is  the  knowl- 
edge of  these  properties  that  has  enabled  us  to  produce  concen- 
trates and  conduct  tests.  Another  practical  consideration 
involved  in  this  matter  of  properties  lies  in  the  effect  of  cooking 
and  commercial  methods  of  food  preparation,  for  not  only  must 
we  learn  where  the  vitamine  resides  but  how  to  prevent  injury  or 
destruction  in  our  utilization  of  the  source. 

The  properties  of  the  vitamines  may  therefore  be  grouped 
under  two  heads:  first  chemical  properties  and  second  physiolog- 
ical properties. 

I.   CHEMICAL  PROPERTIES  OF  VITAMINE  "A" 

a.  This  dietary  factor's  presence  in  butter  fat  and  egg  yolk  fat 
indicates  its  solubility  in  the  fat  and  it  would  naturally  follow 
that  the  fat  solvents  would  suffice  to  remove  it  with  the  fats 
when  food  sources  are  treated  with  such  a  reagent.  Experience 
has  shown  however  that  while  ether  extraction  applied  to  butter 
or  egg  yolk  removes  the  vitamine  with  the  fat  this  process  fails 
when  it  is  applied  to  vegetable  sources  such  as  cotton  seed,  corn 
germ,  spinach,  lettuce,  etc.  Neither  does  the  cold  or  hot  press 
method  of  oil  extraction  liberate  the  vitamine  with  the  oil.  Recent 
experiments  by  Osborne  and  Mendel,  to  which  we  have  pre- 
viously referred,  have  shown  that  preliminary  treatment  of 
vegetable  sources  with  alcohol  seems  to  loosen  the  bond  between 
the  source  and  the  vitamine  and  that  when  this  binding  is  once 
loosened  subsequent  ether  extraction  will  take  the  vitamine 
out.  That  the  binding  is  not  difficult  to  break  is  shown  by  the 
fact  that  when  vegetables  are  eaten  as  a  source  of  vitamine  the 
body  is  able  to  separate  the  complex.  It  is  further  evident  that 
the  body  does  separate  this  complex  and  stores  it  in  animal  fat 

62 


CHEMICAL   PROPERTIES   OF   VITAMINES  63 

from  the  experiments  with  cow  feeds  and  feeding.  Milk  for 
example  is  rich  or  poor  in  vitamine  according  to  the  supply  of  the 
latter  in  the  food  given  to  the  cow.  The  only  logical  conclusion 
to  be  drawn  from  this  observation  is  that  the  cow  does  not  syn- 
thesize this  factor  but  splits  it  off  from  the  food  source  and  theny 
since  it  is  fat  soluble,  is  able  to  mobilize  it  in  the  butter  fat  of  the 
milk  or  to  a  more  limited  extent  in  the  body  fat.  This  observa- 
tion as  to  the  dependence  of  milk  content  upon  food  has  been 
confirmed  in  the  case  of  nursing  mothers  and  suggests  the  need 
of  especial  attention  to  the  diet  of  the  mother  during  the  lactating 
period. 

b.  It  has  been  generally  assumed  that  the  "A"  vitamine  is 
comparatively  stable  to  heat.  Sherman,  MacLeod  and  Kramer 
state  that  "dry  heating  at  a  temperature  of  100°C.  with  free 
access  of  air,  only  very  slowly  destroyed  fat  soluble  vitamine." 
Osborne  and  Mendel  reported  that  butter  fat  treated  with  steam 
for  two  hours  and  a  half  did  not  appear  to  have  lost  its  value  as  a 
source  of  this  vitamine.  Drummond's  earlier  work  with  fish  oils 
and  whale  oils  seemed  to  confirm  this  conclusion.  Sherman 
and  his  co-workers  cited  above  put  it  this  way:  "The  results 
thus  far  obtained  emphasize  the  importance  of  taking  full  account 
of  the  time  as  well  as  the  temperature  of  heating,  and  of  the  initial 
concentration  of  the  vitamine  in  the  food,  as  well  as  of  the  oppor- 
tunity for  previous  storage  of  the  vitamine  by  the  test  animal." 
More  recent  work  by  Steenbock  and  his  co-workers  in  America 
shows  that  these  earlier  results  are  incorrect  in  the  case  of  butter 
fat  and  that  twelve  hours  exposure  of  butter  fat  to  100°C.  may, 
under  certain  conditions,  destroy  the  efficiency  of  that  substance 
as  a  source  of  the  vitamine.  Drummond  and  other  English 
workers  have  confirmed  Steenbock  in  later  experiments.  Their 
work  has  shown  that  the  presence  or  absence  of  oxygen  is  a  factor 
which  may  determine  the  extent  of  destruction  of  the  vitamine. 
Heat  alone  is  of  very  limited  effect  but  when  sources  are  heated 
in  the  presence  of  oxygen  destruction  of  the  A  vitamine  may  be 
very  rapid.  Drummond  attributes  the  absence  of  the  A  vitamine 
in  lard  to  the  oxidation  that  takes  place  in  the  commercial  ren- 
dering of  this  product.  We  must  conclude  therefore  that  while 
the  vitamine  may  be  destroyed  by  continuous  exposure  to  a  tern- 


64  VITAMINE   MANUAL 

perature  of  100°C.  the  effect  is  largely  determined  by  the  nature 
of  the  process  and  the  way  the  vitamine  is  held  in  the  source. 
Cooking  of  vegetables  therefore  will  not  as  a  rule  result  in  appre- 
ciable destruction  of  this  factor. 

c.  The  process  of  hydrogenation  used  in  hardening  fats  appears 
to  completely  destroy  the  vitamine,  hence  the  many  lard  substi- 
tutes now  in  use  must  in  general  be  considered  "A"  vitamine- 
free  regardless  of  the  content  of  "A"  in  the  fats  from  which  they 
are  derived  unless  they  have  been  made  by  blending  instead  of 
hydrogenation. 

d.  Acids  and  alkalies  have  apparently  little  effect  on  this  par- 
ticular vitamine. 

It  may  be  well  to  state  here  however  that  owing  to  variability 
in  behavior  with  variation  in  conditions  it  is  dangerous  to  draw 
too  general  conclusions  and  until  a  given  source  has  actually 
been  investigated  under  specific  cooking  conditions  one  should 
not  rely  too  strongly  on  analogies  based  on  comparative  experi- 
ments. This  statement  applies  to  all  vitamines  and  presents  one 
of  the  live  subjects  of  investigation  for  the  cooking  schools  and 
the  food  factories. 

e.  Little  has  been  learned  further  about  the  chemistry  of  this 
substance.1  Butter  fat,  nitrogen  free  and  phosphorus  free  is 
shown  to  carry  the  vitamine  and  it  is  therefore  assumed  that  the 
vitamine  lacks  these  elements.    It  has  been  claimed  that  it  may 
be  removed  from  butter  fat  by  prolonged  extraction  with  water 
but  this  has  not  been  confirmed  by  more  recent  experimenters. 
Steenbock  was  the  first  to  call  attention  to  the  association  of  the 
A  vitamine  with  yellow  pigment  in  plant  and  animal  sources. 
Butter,  egg  yolk,  carrots,  yellow  corn  contain  it  while  white  corn 
and  white  roots  are  less  rich  in  this  vitamine.    This  observation 
suggested  the  chemical  relation  between  the  vitamine  and  carotin. 
It  has  however  been  shown  by  Palmer  and  others  that  carotin  is 
not  vitamine  A.     This  association  of  the  pigment  with  the  vita- 
mine  is  therefore  apparently  a  coincidence  and  this  clue  has  failed 
as  yet  to  throw  light  on  the  chemical  nature  of  vitamine  A. 

1  Since  the  above  was  put  in  type  Steenbock  has  shown  that  the  A 
vitamine  resists  saponification  and  that  by  saponifying  fats  which  contain 
the  A  it  may  be  possible  to  secure  a  fraction  rich  in  the  vitamine  and  free 
of  fat. 


CHEMICAL  PROPERTIES   OF  VITAMINES  65 


When  Funk  first  studied  this  substance  he  conducted  all  his 
evaporations  in  vacuo  from  fear  that  higher  temperatures  would 
prove  destructive.  Subsequent  investigation  however  has  shown 
that  100°  has  very  little  if  any  destructive  effect  if  the  vitamine  is 
held  in  acid  or  neutral  solution.  Temperatures  between  100° 
and  120°  maintained  in  an  autoclave  at  15  pounds  above  normal 
pressure  do  tend  to  slowly  destroy  the  factor.  The  extent  of 
this  destruction  also  varies  with  the  character  of  the  crude 
extract.  In  general,  then,  there  is  little  fear  of  injuring  this 
vitamine  in  ordinary  cooking  temperatures  if  the  use  of  alkali  is 
avoided. 

The  effect  of  alkali  depends  upon  the  temperature  to  a  very 
marked  degree.  Osborne  has  recently  reinvestigated  this  matter 
and  finds  that  in  the  presence  of  a  O.lN  solution  of  alkali  at  20°C. 
there  is  very  little  destruction  but  that  raising  the  temperature 
to  90°C.  brings  about  a  marked  destruction.  Seidell  has  shown 
that  if  the  vitamine  is  absorbed  by  Lloyd's  reagent  and  this 
reagent  be  then  extracted  with  dilute  alkali  the  vitamine  passes 
into  the  alkaline  solution.  If  the  latter  is  neutralized  quickly  it  is 
possible  to  recover  most  of  the  vitamine  by  this  method.  The 
effect  of  alkali  becomes  of  practical  importance  to  the  house- 
wife because  of  certain  cooking  habits.  I  refer  to  the  well  known 
practice  of  adding  soda  to  the  water  in  which  vegetables  are 
cooked  to  soften  the  vegetable  and  accelerate  the  cooking. 
Daniels  and  Loughlin  in  this  country  investigated  this  matter 
and  came  to  the  conclusion  that  this  procedure  did  not  produce 
enough  destruction  to  be  dangerous.  Later  the  matter  was 
studied  by  Chick  and  Hume  in  England  and  these  investigators 
brought  out  a  feature  that  had  perhaps  been  overlooked  in  the  pre- 
vious work.  Their  point  was  that  in  ordinary  feeding  tests  the  results 
merely  tell  whether  there  is  enough  vitamine  present  to  produce 
normal  growth.  Hence  if  the  substance  tested  has  much  vita- 
mine,  a  large  part  of  it  might  be  destroyed  and  this  fact  not 
appear  in  the  test  because  enough  might  still  be  left  to  induce 
normal  growth.  By  reducing  the  amount  tested  so  that  it  was 
just  adequate  for  normal  growth  and  then  applying  the  soda- 


66  VITAMINE   MANUAL 

cooking  experimentation  they  showed  that  this  method  of  cook- 
ery does  do  serious  harm  to  the  vitamine.  From  the  practical 
point  of  view  it  is  of  course  sufficient  to  show  that  enough  is  left 
after  a  cooking  process  to  suffice  for  normal  growth  when  the 
substance  is  taken  in  the  portion  sizes  ordinarily  eaten.  The 
effect  of  alkali  deserves  more  attention  on  the  part  of  cooks  and 
food  preparateurs  and  we  need  more  data  concerning  the  minimal 
dose  necessary  to  protect  the  human  animal. 

In  neutral  and  acid  solution  it  is  perfectly  safe  to  assume  little 
destruction  of  this  vitamin  through  heat  and  it  is  now  common 
practice  to  boil  sources  with  the  extracting  reagent  and  to  use 
the  steam  bath  freely  to  concentrate  and  evaporate  these  extracts. 
We  have  recently  investigated  the  effect  upon  cabbage  of  cooking 
in  a  pressure  cooker  at  eight  pounds  pressure.  The  cabbage  so 
cooked,  when  dried  and  mixed  so  as  to  form  10  per  cent  of  a  basal 
vitamine  free  diet,  yielded  all  the  "B"  vitamine  necessary  to 
produce  normal  growth  in  rats. 

The  very  name  of  this  vitamine  indicates  its  ready  solubility 
in  water.  It  is  also  soluble  in  95  per  cent  alcohol  and  either  of 
these  extractants  may  be  used  to  obtain  the  vitamine.  It  is  not 
readily  soluble  in  absolute  alcohol  and  95  per  cent  is  not  as  good 
an  extractant  as  water.  Substances  rich  in  the  vitamine  appar- 
ently yield  the  latter  more  readily  if  they  have  first  been  sub- 
jected to  autolysis  or  if  the  extracting  fluid  is  acidified.  Funk 
was  the  first  to  show  that  yeast  produced  a  greater  yield  if  it 
was  allowed  to  autolyse  before  extraction  with  alcohol.  How- 
ever, Osborne  and  Wakeman  have  produced  a  method  of  treat- 
ing fresh  yeast  by  boiling  it  with  slightly  acidified  water  which 
seem  as  efficient  as  autolysis  in  the  yield  produced. 

The  various  methods  of  extraction  now  in  vogue  have  already 
been  discussed  in  Chapter  II  and  need  not  be  repeated  here. 
In  general  it  is  apparent  that  to  obtain  concentrates  of  high 
potency  it  is  permissible  to  employ  temperatures  of  100°C.  if  we 
will  maintain  an  acid  or  neutral  reaction  but  that  alkali  should 
be  avoided  wherever  possible  and  when  its  use  is  imperative  the 
temperature  must  be  kept  below  20°C.  or  destruction  will  result. 
In  applying  this  rule  to  cooking  operations  the  results  should  be 
determined  by  direct  tests  rather  than  by  assumptions  based  on 


CHEMICAL   PROPERTIES   OF  VITAMINES  67 

these  generalizations.  It  should  also  be  noted  that  the  alkalinity 
of  a  solution  should  be  determined  on  the  basis  of  hydrogen  ion 
concentration  and  not  on  amount  of  alkali  added  since  many 
substances  have  a  marked  buffer  reaction. 

The  water-soluble  "B"  is  not  only  soluble  in  water  but  cnn 
be  dissolved  in  other  reagents.  Thus  McCollum  has  shown  that 
while  benzene  is  of  little  value  as  an  extractant  of  this  vitamine, 
if  we  will  first  extract  the  vitamine  with  alcohol  or  water  and 
deposit  this  on  dextrin  by  evaporation  it  is  then  possible  by 
shaking  the  activated  dextrin  with  benzene  to  cause  the  vitamine 
to  pass  into  solution  in  benzene.  Voegtlin  and  Meyers  have 
recently  shown  that  it  is  soluble  in  olive  oil  and  in  oleic  acid  and 
their  data  suggest  a  new  means  of  concentrating  the  substance 
which  may  be  of  value  in  tracing  its  character. 

The  "B"  vitamine  is  relatively  easily  absorbed  by  finely 
divided  precipitates.  We  have  already  referred  to  the  use  of 
fuller 's  earth  for  this  purpose  by  Seidell.  This  adsorptive  power 
sometimes  manifests  itself  in  the  treatment  of  plant  extracts. 
A  watery  extract  of  alfalfa  can  be  made  to  throw  down  its  protein 
complex  by  diluting  it  to  40  per  cent  with  alcohol.  Osborne 
reports  however  that  this  process  frequently  removes  the  vita- 
mine  also  which  appears  to  be  thrown  down  with  the  precipi- 
tated material.  This  adsorptive  power  therefore  often  appears 
as  a  difficulty  in  the  handling  of  the  substance  as  well  as  a  means 
of  extraction.  We  have  used  Osborne 's  method  with  alfalfa 
extracts  and  find  the  above  result  is  not  by  any  means  invariable, 
for  in  some  of  our  extracts  we  retained  the  greater  part  of  the 
vitamine.  Kaolin  and  ordinary  charcoal  are  not  very  good 
adsorbents  but  the  latter  can  be  activated  to  serve  this  purpose. 

The  elemientary  nature  of  the  "B"  vitamine  remains  a  mystery. 
Extracts  which  contain  it  show  the  presence  of  nitrogen.  Funk's 
earlier  researches-  on  yeast  and  rice  polishings  both  yielded 
crystalline  complexes  which  he  analysed.  His  data  on  this  sub- 
ject follow: 

A.  The  yeast  complex 

Crystals  melting  at  233°C.  consisting  of: 

I.  A  complex  melting  at  229 °C.  and  forming  needles  and  prisms 
nearly  insoluble  in  water  and  with  the  apparent  formula  of 
C24HI909N8. 


68  VITAMINE   MANUAL 

II.  A  complex  melting  at  222°C.   and  soluble  in  water.    Formula 

Ca9H2,09N6. 
III.  Nicotinic  acid  melting  at  235°C.    CaH6O2N. 

B.  The  rice  complex 

Crystals  melting  at  233°C.  consisting  of: 

I.  A  complex  melting  at  233°G.  and  with  a  formula  of  CzaHjoC^Ni. 
II.  Nicotinic  acid  melting  at  235°C.    C6H5O8N. 

Funk  held  at  the  time  that  the  possible  nature  of  the  compound 
was: 

T 

OC 


HIST 

It  was  this  idea  that  led  him  to  call  it  an  "amine." 

We  are  unable  at  present  to  report  any  nearer  approach  to  the 
elementary  analysis  and  all  attempts  at  purification  have  shown 
a  tendency  to  make  the  active  substance  either  disappear  entirely 
or  else  distribute  itself  over  the  several  fractions  instead  of  con- 
centrating itself  in  one.  Its  basic  nature  seems  to  be  well  estab- 
lished by  its  behavior  with  phosphotungstic  acid  and  its  ready 
adsorption  by  carbons  activated  to  take  up  basic  substances. 

III.   THE     CHEMICAL     PROPERTIES     OF     WATER-SOLUBLE     "c" 

The  properties  of  this  newest  member  of  the  family  are  still 
less  defined.  All  are  agreed  that  it  is  much  more  sensitive  to 
heat  and  alkali  than  the  other  two.  Temperatures  above  50°C. 
are  usually  destructive  though  the  time  factor  is  extremely  impor- 
tant as  well  as  the  reaction.  Hess  for  example  has  found  that 
the  temperature  used  to  pasteurize  milk  continued  for  some 
time,  is  more  destructive  to  the  vitamine  than  boiling  water 
temperature  continued  for  only  a  few  minutes.  The  extent  to 
which  orange  juice  and  tomato  juice  will  resist  high  temperatures 
indicates  the  protective  action  of  acids  to  be  considerable. 

Dr.  Delf's  experiments  at  the  Lister  Institute  were  especially 
directed  to  the  behavior  of  this  vitamine  in  cabbage.  She  first 


CHEMICAL  PROPERTIES   OF  VITAMINES  69 

determined  the  minimum  dose  of  raw  cabbage  required  to  pre- 
vent scurvy  in  guinea  pigs  and  found  that  it  was  less  than  1.5  grams 
and  more  than  0.5  gram  daily.  When  the  cabbage  was  heated 
in  water  at  60°C.  for  an  hour,  symptoms  of  severe  scurvy  were 
just  prevented  by  5  grams  of  the  cooked  cabbage  fed  daily.  By 
heating  at  70°,  80°,  90°  and  100°  for  the  same  length  of  time  the 
5  grams  of  cooked  material  could  be  made  non-effective  as  a  pre- 
ventive. Her  conclusions  are  that  when  cabbage  is  cooked  for 
one  hour  at  temperatures  ranging  from  80°  to  100°C.  the  cabbage 
leaves  lose  about  90  per  cent  of  the  antiscorbutic  power  originally 
held  by  the  raw  equivalent.  Sixty  minutes  at  60°  or  twenty 
minutes  at  90°  to  100°  resulted  in  about  80  per  cent  destruction. 
Dr.  Delf  calls  attention  also  to  the  fact  that  the  effect  of  the  heat 
is  increased  to  only  a  slight  degree  by  rise  in  temperature.  Assum- 
ing that  the  effect  of  the  rise  is  orderly,  a  temperature  coefficient 
of  1.3  is  indicated  for  each  rise  of  10°C.  This  low  result  suggests 
to  Delf  a  contradiction  to  any  theory  which  imputes  to  the 
vitamine  enzyme  or  protein-like  qualities  and  on  the  other  hand 
suggests  that  the  substance  is  much  simpler  in  constitution.  Her 
results  also  confirm  Hoist  and  Frohlich  as  showing  its  great  sensi- 
tiveness at  temperatures  of  100°  and  below  and  obviously  have 
a  direct  bearing  upon  cookery  methods. 

The  substance  is  soluble  in  water  and  passes  through  a  parch- 
ment membrane  or  a  porcelain  filter.  Unlike  the  "B"  it  is 
apparently  not  adsorbed  by  fine  precipitates  such  as  fullers' 
earth  or  colloidal  iron.  Harden  and  Zilva  showed  that  when  a 
mixture  of  equal  volumes  of  autolysed  yeast  and  orange  juice  is 
treated  with  fuller's  earth  the  "B"  is  removed  and  the  "C"  left 
unaltered.  Eddy  and  LaMer  have  treated  orange  juice  with 
fullers'  earth  and  then  tested  the  filtered  off  juice  as  cure  and 
preventive  of  scurvy  in  guinea  pigs.  Their  results  showed  that 
6f  cc.  of  the  treated  juice  was  curative,  hence  the  loss  due  to 
adsorption  must  be  less  than  60  per  cent  to  70  per  cent.  Harden 
and  Zilva  were  among  the  first  to  state  that  the  vitamine  is  much 
more  stable  in  acid  than  in  alkali.  They  have  shown  that  even 
1/50  N  sodium  hydrate  at  room  temperature  has  a  rapidly  destruc- 
tive effect.  On  the  other  hand  Delf  showed  that  when  0.5  gm.  citric 
acid  is  added  to  the  water  in  which  germinated  lentils  are  boiled, 


70  VITAMINE   MANUAL 

the  loss  of  the  antiscorbutic  properties  is,  if  anything,  greater 
than  when  no  addition  of  acid  is  made.  She  therefore  concluded 
that  in  cooking  vegetables  there  should  be  no  addition  of  either 
acid  or  alkali  to  the  cooking  water  if  one  wishes  to  conserve  this 
vitamine.  Sherman,  LaMer,  and  Campbell  have  been  engaged 
in  experiments  bearing  on  this  point  throughout  the  past  two 
years.  Some  of  their  results  have  recently  been  published  and 
their  observations  are  worthy  of  special  attention  from  their 
bearing  on  the  character  of  reaction  of  the  vitamine  in  general. 
They  first  proceeded  to  determine  the  amount  of  filtered  tomato 
juice  just  necessary  to  produce  scurvy  in  degrees  extending  from 
no  protection  to  complete  protection  and  they  also  constructed 
a  basal  diet  which  is  apparently  optimum  in  nutrients  and  all 
other  factors  except  the  "C"  vitamine.  They  found  that  at  the 
natural  acidity  of  tomato  juice  (pH  4.2)  boiling  for  one  hour 
destroyed  practically  50  per  cent  of  the  antiscorbutic  power  and 
by  boiling  for  four  hours  they  destroyed  70  per  cent,  which  indi- 
cates that  the  curve  of  the  destructive  process  tends  to  flatten 
more  than  that  of  a  unimolecular  reaction.  This  result  was  con- 
firmed by  heating  experiments  conducted  at  60°,  80°  and 
100°.  In  all  cases  the  temperature  coefficients  are  low.  (Qi0 
equals  1.1-1.3)  confirming  Delfs  results.  When  the  natural 
acidity  of  the  juice  was  first  neutralized  in  whole  or  in  part,  the 
juice  then  boiled  for  an  hour  and  immediately  cooled  and  reacid- 
ified,  it  was  found  that  at  less  than  half  neutralization  (pH  5.1-4.9) 
the  destructive  effect  of  an  hour's  boiling  was  increased  to  58  per 
cent.  When  alkali  was  added  to  an  initial  pH  11  (about  N/40 
titratable  alkali  to  phenolphthalein)  which  fell  to  9  during  the 
hour's  boiling  the  destructive  effect  was  about  65  per  cent.  When 
reacidification  was  omitted  and  the  neutralized  boiled  juice  stored 
in  a  refrigerator  for  five  days  before  using  the  destruction  increased 
90  to  95  per  cent.  These  particular  observations  seem  to  confirm 
the  view  of  Harden  and  Zilva  that  the  vitamine  is  especially 
sensitive  to  alkali.  Hess  has  recently  reported  that  oxygen  is 
destructive  to  this  vitamine. 


CHEMICAL   PROPERTIES   OF  VITAMINES 


71 


IV.   PHYSIOLOGICAL    PROPERTIES    OF   THE    "A"    VITAMINS 

Most  authorities  are  now  agreed  that  both  the  "A"  and  "B;J 
types  are  essential  to  growth.  Rohmann  still  holds  out  against 
the  vitamine  hypothesis.  McCollum  has  recently  pointed  out 
that  while  rats  do  not  have  scurvy  it  does  not  at  all  follow  that 
the  absence  of  the  "C"  in  their  diet  is  immaterial,  but  that  the 
contrary  is  true.  Failure  to  grow,  then,  may  manifest  itself  as  a 
result  of  the  absence  of  either  of  the  first  two  types  and  possibly 
is  affected  by  the  absence  of  the  "C."  We  have  already  seen 
how  this  failure  may  be  utilized  to  measure  the  vitamine  content 
of  a  source.  The  absence  of  the  "A"  type  however  may  also 
manifest  itself  in  another  way,  viz.,  by  the  development  of  an 
eye  disease  which  McCollum  first  designated  as  xerophthalmia  or 
dry  eye  and  which  the  British  authorities  prefer  to  designate  as 
keratomalacia.  The  failure  of  this  result  to  always  follow  the 
absence  of  the  "A"  type  in  the  diet  has  led  some  to  question  the 
specificity  of  this  disease.  While  the  infection  of  the  eye  is  due 
to  other  agents  the  sum  of  the  evidence  supports  McCollum  and 
points  to  the  absence  of  "A"  as  the  true  predisposing  cause  of 
the  disease.  Bulley,  basing  her  claims  on  a  study  of  some  500 
rats  fed  on  a  synthetic  diet,  claims  that  the  eye  condition  is  not 
primarily  due  to  a  dietary  deficiency  but  to  an  infection  resulting 
from  poor  hygienic  conditions.  In  reply  to  her  contentions 
Emmett  has  reviewed  his  own  data  and  presents  them  in  the 
following  summation: 


BAT  GROUPS 

KIND  OP1  VITAMINE 
ABSENT  IN  THE  RATION 

NUMBER  CASES 
REPORTED 

POSITIVE  CASES 
OF  XEROPH- 
THALMIA 

PER  CENT 
POSITIVB1 

A 

Fat-soluble  "A'* 

122 

120 

98 

B 

Water-soluble  "B" 

103 

0 

0 

C 

None 

216 

0 

0 

In  these  groups  special  hygienic  measures  were  taken  against 
infection.  Furthermore  repeated  attempts  were  made  to  trans- 
mit the  eye  disease  by  using  sterile  threads,  passing  them  care- 
fully over  the  edges  of  the  sore  lids  and  then  carfully  inoculating 
the  eyes  of  other  rats.  These  attempts  resulted  negatively  in 


72  VITAMINE  MANUAL 

all  cases  where  the  inoculated  rats  had  plenty  of  the  "A"  vita- 
mine.  Treatment  of  advanced  cases  of  sore  eyes  with  a  saturated 
solution  of  boric  acid  and  also  with  a  silver  protein  solution 
failed  to  relieve  the  condition  while  as  little  as  2  per  cent  of  an 
extract  containing  the  "A"  vitamine  when  added  to  the  ration, 
speedily  resulted  in  cure  and  increase  of  weight.  These  results 
combined  with  similar  data  compiled  by  Osborne  and  Mendel 
seem  to  refute  Bulley's  contentions  and  to  justify  our  acceptance 
of  xeropthalmia  as  a  specific  vitamine  deficiency  disease. 

Osborne  and  Mendel  data 

Total  N.. 

Rats  on  diets  deficient  in  A  vitamine 136  69 

"     on  diets         "         "    B         "        225  0 

"     on  diets  otherwise  deficient 90  0 

"    on     "     experimental  but  probably  adequate  201  0 

"    on  mixed  food 348  0 

Totals 1000  69 

On  the  other  hand  all  workers  know  that  rats  often  do  develop 
and  grow  well  for  a  considerable  period  of  time  on  a  diet  free  from 
the  "A"  and  without  manifesting  the  eye  disease.  The  British 
authorities  explain  this  by  assuming  that  animals  have  the  power 
to  lay  down  a  reserve  of  this  vitamine  on  which  they  can  draw  in 
emergency.  Sherman  and  his  coworkers  confirm  this  power  to 
store  the  vitamine.  Others  have  been  led  to  explain  their  results 
as  due  to  contamination  of  the  basal  diet.  Daniels  and  Loughlin 
recently  maintained  that  the  commercial  lard  used  in  basal  diets 
and  assumed  to  be  "A"  vitamine-free  was  supplied  with  suffi- 
cient of  the  "A"  to  produce  growth  and  prevent  eye  disease. 
Their  views  have  failed  of  confirmation  by  Osborne  and  Mendel. 
It  is  evident  therefore  that  these  occasional  lapses  from  specific 
response  to  absence  of  the  "A"  vitamine  need  further  elucidation. 
It  is  equally  manifest  that  in  the  majority  of  cases  the  absence 
of  the  "A"  will  result  in  both  stunted  growth  and  xeropthalmia. 
The  appearance  of  the  eye  disease  may  be  taken  however,  as  a 
sure  indication  of  the  absence  or  deficiency  in  the  "A"  vitamine. 


CHEMICAL   PROPERTIES   OF  VITAMINES 


73 


V.   PHYSIOLOGICAL  PROPERTIES   OP  THE   "B"    VITAMINE 

Beri-beri  is  a  disease  that  is  described  clinically  as  a  form  of 
severe  peripheral  neuritis  and  may  appear  in  two  well  marked 
forms.  In  one  type  there  is  great  wasting,  anesthesia  of  the  skin 
and  finally  paralysis  of  the  limbs.  In  the  other,  the  most  marked 
symptom  is  excessive  edema  which  may  affect  trunk,  limbs  and 
extremities.  In  severe  cases  the  heart  is  usually  involved  and 
death  may  occur  suddenly  from  heart  failure. 

Most  observers  assume  that  the  antineuritic  vitamine  dis- 
covered by  Funk  and  the  water-soluble  "B"  are  identical.  This 
view  is  based  on  the  fact  that  when  sources  which  yield  the  water- 
soluble  "B"  in  rat  feeding  are  tested  for  antineuritic  power  these 
sources  are  apparently  parallel  in  antineuritic  power  and  growth 
production.  Furthermore  rats  deprived  of  the  water-soluble  "B" 
develop  polyneuroses  identical  in  symptoms  with  those  shown  by 
rats  and  pigeons  when  the  latter  are  placed  on  a  polished  rice 
diet.  The  British  Medical  Board  has  compiled  the  following 
table  to  support  this  view: 


Table  compiled  from  pages  85  and  86,  British  Medical  Research  Committee 

Report 


FOODSTUFF 

VALUE  AS  A  SOURCE  OF 
WATER-SOLUBLE  "B" 
(SHOWN  BY  EXPERI- 
MENTS WITH  RATS) 

VALUE  AS  SOURCE  OF 
THE  ANTINEUHITIC 
FACTOR  OR  ANTI-BERI- 
BERI FACTOR  (SHOWM 
BT  EXPERIMENTS 
WITH  BIRDS) 

Rice  germ    

4-4.4. 

4-4-4-4- 

Wheat  germ       

4-4-4- 

4-4-4- 

Yeast       

4-+4- 

4.4-4- 

Effff  volk 

4-4- 

4-4-4- 

4-4- 

4-4-4- 

Wheat  bran  

4- 

+  + 

4- 

+ 

Milk  

+ 

Slight 

4- 

4- 

Meat  extract  

0 

0 

White  bread  or  flour                 

o 

0 

Polished  rice  

0 

0 

74 


VITAMINE   MANUAL 


BEHAVIOR 

WATEB-SOLUBLB  "fi" 

ANTINEUBITIC  VITAMINE 

Solubility  in  water  

Very  soluble 

Very  soluble 

Solubility  in  alcohol,  dilute 
Solubility  in  absolute  alco- 
hol   

Very  soluble 
Insoluble 

Very  soluble 
Insoluble 

Solubility  in  ether,  chloro- 
form and  benzene 

Insoluble 

Unusually  insoluble 

Stability  to  heat  

Stable  at  100  °C    de- 

but can  be  extract- 
ed with  ether  from 
fatty  materials  such 
as  egg  yolk 
Destroyed  very  slowly 

Stability  to  drying  

stroyed  rapidly  at 
120°  (in  neutral  or 
acid  solution) 

Stable 

at  temperatures  be- 
low   100°C.,    more 
rapid    at    tempera- 
tures  between    110 
and  120°  C. 
Stable 

Stability  to  acids  (hot  di- 
lute)   

Moderately  stable 

Stable 

Stability  to  acids  (cold  di- 
lute).   . 

Stable 

Stable 

Stability  to   alkalies    (hot 
dilute)         

Rapidly  destroyed 

9 

Stability  to  alkalies  (cold 
dilute)     

Stable 

In  dialysis  

Passes  through  parch- 

Passes through  parch- 

In adsorption       

ment  membrane 
Adsorbed  from   acid 

ment  membrane 
Adsorbed    from    neu- 

or neutral  solution 
by    fuller's    earth, 
charcoal,  etc. 

tral     solutions     by 
fuller's    earth,    col- 
loidal ferric  hydrox- 
ide,    animal     char- 
coal, etc. 

Emmett  has  recently  opposed  this  view  and  suggests  that 
while  the  antineuritic  factor  and  the  growth  factor  are  found  in 
the  same  sources  and  have  much  in  common  it  does  not  follow 
that  they  are  identical  and  that  his  experiments  tend  to  show 
that  there  are  marked  differences  which  suggest  that  the  "B" 
type  is  not  a  single  entity  but  a  group.  Mitchell  has  summarized 
very  well  the  controversial  phases  of  this  question  with  an  impar- 
tial review  of  the  facts.  One  of  strongest  of  the  opposition  argu- 


CHEMICAL  PROPERTIES   OF  VITAMINES  75 

ments  lies  in  the  failure  of  milk  to  cure  beri-beri  except  when 
administered  in  large  quantities.  This  objection  has  been  partly 
allayed  by  data  bearing  on  the  relation  of  the  milk  content  to  the 
food  of  the  cow.  Hess,  Dutcher,  Hart  and  Steenbock  and  others 
have  adduced  sufficient  evidence  to  show  that  the  vitamine  con- 
tent of  the  milk  of  a  cow  is  largely  determined  by  the  cow's  food 
and  as  a  consequence  the  milk  may  be  very  poor  in  vitamine.  It 
is  obvious  then  that  the  failure  of  the  milk  to  cure  beri-beri  in  a 
given  case  might  be  due  to  this  cause  and  not  to  lack  of  identity 
of  the  curative  with  the  growth  factor.  Osborne  and  Mendel 
have  also  shown  that  milk  in  general  must  not  be  classed  among 
the  rich  sources  of  the  vitamine,  even  when  the  cow's  food  is 
rich  in  vitamine.  The  principal  facts  in  the  controversy  have 
been  presented  and  at  present  the  evidence  for  regarding  the 
vitamines  identical  seems  to  be  preponderant. 

Recently  Auguste  Lumiere  in  Paris  has  put  forth  the  view 
that  polyneuritis  is  not  merely  a  vitamine  deficiency  disease 
but  a  nutriment  deficiency  disease.  He  reports  that  he  fed 
birds  on  a  starvation  diet,  but  with  plenty  of  vitamine  "B." 
These  birds  developed  polyneuritis  and  were  cured  by  adding  to 
the  diet  plenty  of  polished  rice.  The  view  he  wishes  us  to  take 
is  that  all  factors  must  be  present  and  that  the  absence  of  the 
nutriment  is  as  important  as  the  absence  of  the  vitamine. 

In  the  field  of  nutrition  the  absence  of  the  "B"  type  is  partic- 
ularly marked  by  the  behavior  of  the  deprived  animal.  Rats 
transferred  from  a  vitamine-free  diet  to  one  containing  the 
"B"  only,  make  a  much  more  rapid  recovery  toward  normal 
(even  in  the  absence  of  the  "A")  than  do  animals  transferred 
from  the  vitamine-free  diet  to  one  containing  the  "A"  and  not 
the  "B."  This  initial  jump  from  addition  of  the  "B"  will  not 
continue  long  in  the  absence  of  the  "A,"  as  a  general  rule.  Hess 
believes  that  in  some  of  his  infants  he  was  able  to  show  markedly 
successful  growth  on  the  diet  deficient  in  the  "A"  but  rich  in  the 
"B."  It  is  not  certain  however  that  his  diets  were  sufficiently 
devoid  of  the  "A"  factor  to  be  declared  "A"  vitamine-free  and 
we  know  little  of  the  amount  of  the  "A"  necessary  to  normal 
infant  growth.  All  results  however  show  that  both  "  A"  and  "  B" 
are  necessary  to  growth  production  and  though  the  term  growth 


76  VITAMINE   MANUAL 

vitamine  was  applied  to  the  "A"  originally  the  distinction  is 
one  that  should  be  rejected,  for  both  "A"  and  "B"  and  possibly 
"C"  are  all  entitled  to  this  name. 

The  manner  in  which  the  "B"  vitamine  acts  is  still  obscure. 
Voegtlin  some  time  ago  tried  to  demonstrate  that  it  was  identical 
with  secretin  and  stimulated  pancreatic  flow.  Recent  work  at 
the  Johns  Hopkins  University  by  Cowgill  and  by  Aurep  and 
Drummond  in  England  has  failed  to  confirm  this.  One  of  its 
most  marked  immediate  effects  is  increase  in  appetite.  Karr 
in  Mendel's  laboratory  has  shown  that  dogs  which  refused 
their  basal  diet  would  resume  eating  it  if  they  were  allowed  to 
ingest  separately  a  little  dried  yeast.  Karr  studied  the  metab- 
olism of  these  dogs  as  regards  nitrogen  partition  but  the  results 
give  little  data  that  is  explicatory  of  the  behavior  of  the  vita- 
mine.  In  1915  the  author  was  able  to  bring  about  marked  imme- 
diate improvement  and  the  ultimate  recovery  of  a  number  of 
infants  who  were  of  the  marasmic  type  by  merely  increasing 
the  "B"  vitamine  content  of  their  food.  In  these  cases  the 
vitamine  was  carried  by  Lloyd's  reagent  and  administered 
mixed  with  cereal,  or  the  crude  extract  was  combined  with 
the  milk.  The  pancreas  of  the  sheep  was  the  source  used. 
In  these  cases  the  growth  curve  changed  abruptly  from  a 
decline  to  a  sharp  rise  and  this  increase  in  weight  continued  and 
was  accompanied  by  all  the  other  signs  of  improved  nutrition 
including  increase  in  appetite.  The  change  in  the  growth  curve 
from  decline  to  rise  was  accomplished  without  increasing  or  chang- 
ing the  basal  diet  but  as  the  appetite  increased  the  food  had  natu- 
rally to  be  increased  to  keep  pace.  In  these  cases  the  effect  of 
the  vitamine  was  to  enable  the  child  to  utilize  its  normal  food 
and  to  increase  its  appetite  for  it.  This  action  certainly  suggests 
stimulation  of  digestive  glands.  It  also  showed  that  even  though 
the  diet  may  contain  the  vitamine  as  was  the  case  in  the  milk  fed 
to  these  children  the  addition  of  the  vitamine  in  concentrated 
form  often  gives  an  upward  push  that  the  food  mixture  fails  to 
accomplish.  Daniels  and  Byfield  have  recently  confirmed  the 
effect  of  increased  "B"  in  infant  growth.  Cramer  has  suggested 
in  a  paper  published  recently  in  The  American  Journal  of  Physi- 
ology that  the  fatty  tissue  about  the  suprarenals  may  be  a  deposi- 


JOHNG. 


FIG.  8.  THE  EFFECT  OF  VITAMINE  B  ON  A  MARASMIC  INFANT 

1.  On  the  twentieth  day  the  patient  developed  a  cough.  2.  On  the 
twenty -first  day  the  cereal  was  reduced  from  three  times  a  day  to  twice  a 
day.  The  patient  cried  during  the  night.  8.  On  the  twenty-second  day 
the  stools  showed  free  starch.  4-  On  the  twenty-third  day  an  anal  abscess 
was  opened.  The  stools  continued  to  show  free  starch  until  the  twenty- 
fifth  day.  5.  On  the  twenty-fifth  day  the  stools  showed  soluble  starch 
but  no  free  starch.  6.  On  the  twenty -seventh  day  the  appetite  was  good 
and  there  was  no  starch.  7.  From  the  twenty-eighth  to  the  forty-third 
day  no  starch  was  observed  in  the  stools.  8.  On  the  thirty-first  day  the 
patient  developed  a  cough.  9.  From  the  forty-ninth  day  to  the  time  of 
discharge  three  tablespoonsful  of  orange  juice  were  given  daily.  10.  On 
the  seventy-third  day  the  patient  developed  a  bronchitis  and  mustard 
paste  was  applied  every  four  hours  up  to  the  eighty-fourth  day. 

F1  =  From  the  twenty-first  day  to  the  forty-third  day  the  patient 
received  each  day  2  grams  of  Lloyd  powder,  activated  with  pancreatic 
vitamin.  The  powder  was  administered  by  mixing  1  gram,  with  each  cereal 
feeding.  The  result  was  20  ounces  gain  in  twenty-two  days,  a  normal 
growth. 

F2  =  After  a  period  of  ten  days  without  vitamin,  during  which  the 
patient  settled  down  to  a  level  growth  curve,  the  treatment  described 
under  V1  was  resumed.  This  was  continued  from  the  fifty-third  to  the 
seventy-sixth  day.  The  result  was  the  resumption  of  growth  but  at  a 
slower  rate;  8  ounces  were  gained  in  twenty-three  days.  During  the  latter 
part  of  the  period  the  patient  developed  a  bronchitis.  At  the  end  of  this 
period  the  patient  was  placed  on  a  whole  milk  formula.  From  that  time 
to  the  time  of  discharge  the  patient  grew  normally. — From  the  American 
Journal  of  Diseases  of  Children,  1917,  xiv,  189. 

77 


78 


VITAMINE   MANUAL 


tory  of  vitamine  and  that  in  the  absence  of  vitamine  this  tissue 
loses  its  supply  and  that  this  is  the  explanation  of  lessened  activity 

Effecfts  of  Vitamines  on  Growth 


indiealw 


Rat  16 


Djys 


FIG.  9 

of  that  gland  in  certain  metabolic  disturbances.     This  idea  tends 
to  support  the  idea  that  vitamines  are  gland  stimulants  or  hormones 


CHEMICAL  PROPERTIES   OF  VITAMINES  79 

and  the  word  food  hormone  has  been  suggested  to  describe  them 
on  that  account.  A  few  years  ago  Calkins  and  Eddy  tried  to 
determine  the  effect  of  the  vitamine  on  the  single  cell  by  use  of 
the  paramecium  but  the  results  of  the  experiments  failed  to  show 
a  vitamine  requirement  on  the  part  of  these  animals.  McDougall 
has  recently  suggested  that  the  vitamines  produce  their  effect  on 
yeast  cells  by  increasing  hydration.  Unfortunately  nearly  all 
stimuli  which  produce  growth  are  accompanied  by  hydration 
effects  and  it  is  difficult  to  feel  that  this  is  a  specific  vitamine 
effect  although  without  denying  the  possibility.  Dutcher  has 
tried  to  show  that  vitamines  have  a  relation  to  oxidation  effects. 
He  observed  that  the  issues  of  polyneuritic  birds  showed  a  marked 
reduction  in  catalase  and  that  this  catalase  was  restorable  by  cur- 
ing the  birds  with  vitamine.  The  main  difficulty  lies  in  the 
conflexity  of  factors  that  function  between  cause  and  effect. 

These  views  are  at  best  speculations.  The  literature  is  singu- 
larly lacking  in  detailed  metabolic  analyses  of  excreta  of  animals 
during  vitamine  stimulation  and  we  know  nothing  of  the  possi- 
bilities of  overdosage,  for  in  all  the  work  done  it  has  been  gener- 
ally assumed  that  the  presence  of  an  amount  greater  than  that 
necessary  to  produce  normal  growth  is  not  material. 

The  exact  manner  of  the  vitamine's  action  then  remains  to  be 
determined  and  it  is  obvious  that  this  solution  will  come  much 
more  rapidly  if  we  can  first  identify  the  substance  chemically. 

VI.   THE    PHYSIOLOGICAL   PROPERTIES    OP   THE    "c"    VITAMINE 

The  steps  that  led  to  the  acceptance  of  scurvy  as  a  vitamine 
deficiency  disease  have  already  been  discussed  and  show  how  the 
vitamine  acts  in  such  a  disease.  Practically  all  the  work  done 
with  this  vitamine  to  date  has  been  concerned  either  with  dosage 
or  with  reaction  to  heat,  drying,  etc.  The  only  paper  that  we 
have  seen  that  suggests  another  function  than  antiscorbutic 
power  for  this  vitamine  is  the  one  by  McCollum  and  Parsons  in 
which  they  suggest  that  even  in  animals  where  scurvy  does  not 
exist,  the  presence  of  this  factor  may  be  necessary  to  normal 
metabolism.  The  following  table  gives  some  of  the  data  com- 
piled by  the  British  workers  as  to  the  antiscorbutic  power  of 
various  sources: 


80 


VITAMINE   MANUAL 


Table  compiled  from  page  44,  British  Medical  Research  Committee  Report 


FOODSTUFF 

VALUE  AGAINST 
SCURVT 

MINIMUM  DAILY 
RATION  NECESSARY 
TO  PREVENT  SCURVY 
IN  GUINEA  PIGS 

Cereals: 
Whole  grains                     ...       .   . 

0 

o 

o 

0 

Pulses: 
Whole  dry          

o 

Germinated  (lentils)  

4-4- 

5  0  grams 

Vegetables: 
Cabbage  (raw)               

++++ 

1  0  gram 

Cabbage  (cooked  one-half  hour  at 

ioo°c  )        

++ 

5  0  grams 

Runner  beans  (green  pods) 

+++ 

5  0  grams 

Carrot  (juice)    .         

+ 

20.0  cc. 

Beet  root  (juice) 

-j- 

More  than  20  cc 

+++ 

2.5  cc. 

Potatoes  (cooked  one-half  hour  at 
100°C.)  

+ 

20.0  grams 

+ 

Desiccated  vegetables  

0  to  + 

60.0   grams   ex- 

Fruits: 
Lemon  juice  (fresh)           

++++ 

pressed        as 
equivalent  in 
fresh  cabbage 

1.5  cc. 

++ 

5.0  cc. 

Orange  juice  (fresh)           

++++ 

1.5  cc. 

Lime  juice  (fresh)  

-f-f 

10.0  cc. 

Lime  juice  (preserved)  

0  to  + 

Less  than  + 

More  than  20.0 

Apples     .          

Less  than  + 

grams 

Apples  dried           ... 

Less  than  -j- 

Less  than  -f- 

Mango         .   .       

Less  than  -f- 

Less  than  + 

Meat: 
Raw,  juice                

Less  than  -f- 

More  than  20  cc. 

Tinned  

0 

CHEMICAL   PROPERTIES   OF  VITAMINES  81 

A  glance  at  this  table  shows  the  richest  sources  (see  also  table 
on  page  59.)  To  these  must  be  added  canned  tomato  juice 
which  Hess  has  shown  practically  equal  to  orange  juice  in  effi- 
ciency and  uses  with  infants  in  the  same  quantity.  This  dis- 
covery is  of  great  value  in  instances  where  the  cost  of  orange  juice 
is  often  prohibitive. 

LaMer  and  Campbell  have  presented  some  evidence  to  show 
that  the  antiscorbutic  vitamine  has  a  direct  effect  upon  the 
adrenal  glands.  In  their  scurvy  cases  they  find  definite  evidence 
of  the  enlargement  or  hypertrophy  of  this  organ.  Whether  it 
affects  other  organs  or  not  it  remains  to  be  shown. 


CHAPTER  VII 
How  TO  UTILIZE  THE  VITAMINE  IN  DIETS 

In  the  preceding  chapters  it  has  been  the  aim  to  present  the 
findings  of  the  principal  workers  in  the  field.  In  attempting  to 
summarize  the  work  of  so  widely  scattered  a  group  as  are  now 
engaged  in  vitamine  research  it  is  impossible  to  cover  completely 
the  many  investigations  and  it  is  inevitable  that  some  work  will 
have  been  overlooked,  but  the  foregoing  covers  at  least  the  prin- 
cipal data  on  the  subject.  What  is  the  bearing  of  all  this  infor- 
mation on  human  behavior  and  what  lessons  can  the  layman 
draw  from  it  that  is  of  direct  application  to  him?  Let  us  first 
consider  this  question  from  the  dietary  viewpoint. 

I.   INFANT   NUTRITION 

The  limited  character  of  the  infant's  diet  has  made  the  con- 
sideration of  vitamine  content  in  his  diet  much  more  important 
than  in  the  case  of  the  adult  with  the  latter's  wide  variety  of 
choice.  It  is  evident  from  the  previous  data  that  a  growing  infant 
must  not  only  be  provided  with  a  sufficient  supply  of  calories, 
nutrients  and  salts,  but  must  also  have  a  liberal  supply  of  the 
three  vitamines.  Milk  has  in  general  been  classed  as  adequate 
in  all  these  features,  but  the  vitamine  researches  have  forced  us 
to  reconsider  our  views  in  regard  to  this  staple. 

The  first  point  to  be  borne  in  mind  is  that  the  vitamine  content 
of  either  cow  or  human  milk  is  dependent  primarily  upon  the 
food  eaten  by  the  producer  of  the  milk.  In  other  words  milk  is 
merely  a  mobilization  of  the  vitamines  eaten  and  if  the  diet  is  to 
yield  vitamine-rich  milk  it  must  itself  be  rich  in  these  factors. 
Many  a  cow  produces  milk  low  in  vitamine  content  and  the  same  is 
true  of  nursing  mothers.  There  are  many  "old  wives"  prejudices 
in  regard  to  what  food  a  lactating  mother  may  eat  and  unfor- 
tunately many  of  these  prejudices  are  extremely  injurious  and 
false.  One  of  them  is  the  prejudice  against  green  vegetables. 
Experience  has  shown  that  under  ordinary  conditions  such 

82 


THE   VITAMINES   IN   DIETS  83 

vegetables  are  well  tolerated  by  the  mother  and  from  their  con- 
tent of  vitamine  it  is  evident  that  they  are  suppliers  of  these 
factors.  In  the  case  of  the  cow  the  fact  that  cereals  are  poor  in 
some  of  the  vitamines  and  green  grasses  rich  therein,  teaches  a 
lesson  that  bears  directly  upon  winter  feeding  of  cattle  if  the  milk 
supply  is  to  be  used  for  infants.  We  need  a  series  of  diets  and 
cattle  foods  for  just  this  purpose  of  insuring  the  proper  vitamine 
content  in  milk.  The  preceding  tables  will  enable  one  to  develop 
such  diets  fairly  satisfactorily,  but  more  data  is  urgently  needed. 

The  second  point  in  regard  to  milk  lies  in  the  effect  of  pas- 
teurization. This  measure  is  now  well  nigh  universal  and  in 
America  at  least  has  played  a  tremendous  part  in  the  reduction  of 
infant  mortality,  especially  during  the  summer  months.  At 
present,  however,  we  know  that  this  treatment  while  removing 
dangerous  germs  may  also  eliminate  the  antiscorbutic  factor. 
The  sensible  attitude  then  is  to  recognize  this  fact  and  if  a  clean 
whole  milk  is  not  available  retain  the  pasteurization  and  meet 
the  vitamine  deficiency  by  other  agents.  Such  agents  are  orange 
juice  and  tomato  juice  and  experience  has  already  shown  that 
these  juices  can  be  well  tolerated  by  infants  much  earlier  than 
used  to  be  thought  possible. 

While  the  pasteurization  does  not  appreciably  affect  the  con- 
tent of  "A"  or  "B"  vitamines,  the  variability  in  content  of  these 
vitamines  in  milk  indicates  that  it  may  at  times  be  necessary 
to  supplement  them  in  the  diet.  In  this  connection  it  must  be 
borne  in  mind  that  cereals  vary  widely  in  content  and  cannot  be, 
as  they  often  are  now,  considered  equivalent  in  growth  stimula- 
tion power.  This  is  a  subject  that  needs  special  attention  on 
the  part  of  vitamine  experts  and  dietitians  and  finally  by  the 
food  manufacturers.  A  good  vitamine-rich  cereal  combination 
would  form  an  excellent  adjuvant  to  infant  dietaries  after  they 
reach  the  age  of  tolerance  to  such  a  diet.  But  even  before  that 
time  the  expressed  juice  of  various  vegetables  as  well  as  fruits  is 
found  to  be  well  tolerated  when  mixed  with  the  milk  or  given 
separately,  and  carrot  and  spinach  juice  are  now  being  used  in  this 
connection  with  good  results.  These  juices  like  orange  juice 
contain  the  B  type  in  abundance  and  there  is  no  doubt  that  in 
their  stimulation  to  the  appetite  they  play  an  important  part  in 
making  the  desirable  daily  gain. 


84  VITAMINE   MANUAL 

Fortunately  for  the  layman  he  has  in  the  scales  a  good  indicator 
of  the  normal  progress  of  his  child  and  so  long  as  growth  is  nor- 
mal he  can  fairly  assume  that  the  diet  is  adequate  but  if  the 
scales  say  otherwise  it  is  time  for  him  to  seek  advice  and  then  he 
is  wise  who  insures  that  his  medical  adviser  knows  the  newer 
aspects  of  nutrition.  The  parent  can  do  this  only  by  proper 
selection,  but  with  a  little  knowledge  he  can  soon  satisfy  himself 
as  to  whether  his  pediatrist  is  the  right  sort  and  it  is  one  of  the 
purposes  of  this  text  to  bring  home  to  the  layman  his  responsi- 
bility in  this  matter. 

There  has  grown  up  in  this  country  a  great  regard  for  prepared 
milk  substitutes  in  infant  feeding  and  a  wide  usage  of  condensed 
milks,  reinforced  milks,  diluted  milk  formulae,  etc.  All  such 
preparations  must  be  examined  anew  in  the  light  of  the  vitamine 
discoveries  and  unless  the  given  preparation  can  show  a  clean 
bill  of  health  in  vitamine  content,  it  should  be  either  discarded 
or  properly  supplemented. 

As  children  grow  up,  it  is  fortunate  that  in  their  wider  choice 
of  dietaries  the  danger  of  vitamine  deficiency  decreases.  But 
even  in  childhood  it  is  unsafe  to  rely  too  much  on  chance.  In 
this  country  there  are  well  deserving  movements  on  foot  to  attract 
the  parents  of  the  community  to  the  necessity  of  attention  to 
simple  standards  of  growth  progress,  and  clinics  for  this  purpose 
are  appearing  in  increasing  numbers  with  each  year.  Such 
movements  are  to  be  most  heartily  approved.  It  is  also  possible 
in  these  measures  to  not  only  build  better  children,  but  to  make 
the  children  themselves  intelligent  in  their  rejection  of  unsuitable 
combinations  and  in  that  way  not  only  conserve  their  own  health, 
but  provide  an  educated  body  of  citizens  to  pass  on  the  knowl- 
edge to  future  generations.  In  a  school  in  New  York  City  I 
recently  had  occasion  to  discuss  the  school  lunch  room  and  its 
offerings  with  the  children  of  the  school  in  the  light  of  vitamine 
discoveries.  The  keeness  and  intelligence  shown  by  the  children 
in  the  discussion  that  followed  has  convinced  me  that  in  this 
matter  of  vitamines  the  children  themselves  can  be  relied  upon 
to  assist  materially  in  the  matter  of  better  food  combinations  and 
intelligent  selection. 

Finally  it  must  be  noted  that  one  of  the  most  common  of  infant 
deficiencies  is  the  failure  of  the  bones  to  lay  down  lime.  The 


THE   VITAMINES   IN   DIETS  85 

effect  of  this  failure  is  commonly  described  as  rickets.  The 
British  workers  consider  that  this  deficiency  is  a  lack  of  vitamine 
"A."  Their  views  have  been  set  forth  at  greatest  length  by 
Mellanby,  the  principal  worker  in  this  subject.  While  this  view 
is  still  debatable  and  in  this  country  it  is  not  yet  accepted,  one 
fact  has  come  out  in  the  controversy  and  that  is  the  remarkable 
value  of  cod-liver  oil  as  a  preventive  of  rickets.  It  may  be  that 
the  power  of  the  oil  is  due  to  its  "A"  vitamine  content  in  which 
it  is  known  to  be  rich,  or  it  may  be  due  to  a  new  vitamine,  but 
the  fact  that  the  oil  is  a  preventive  in  this  respect  gives  the 
pediatrist  another  agent  to  insure  normal  growth.  The  various 
views  on  the  causes  of  rickets  are  set  forth  more  in  detail  in 
Chapter  VIII. 

II.   ADULT  DIETS 

A  study  of  the  dietary  habits  of  various  sections  of  the  United 
States  shows  that  there  is  a  very  general  tendency  on  the  part  of 
the  majority  of  the  people  to  confine  their  foods  to  a  meat,  potato, 
and  cereal  diet.  The  use  of  salads  is  looked  upon  by  many  sec- 
tions as  a  foreign  affectation  and  too  little  attention  is  paid  to  the 
value  of  eggs,  milk  and  cheese.  Enough  has  been  said  already 
to  show  that  these  latter  articles  have  much  more  than  an  esthetic 
value  and  one  of  the  missions  of  the  nutrition  expert  must  be  to 
show  the  people  why  dairy  products  and  salads  must  become 
features  in  the  e very-day  meals  of  the  e very-day  people.  And 
even  if  the  salads  are  still  unappreciated,  it  is  necessary  that 
cooked  green  vegetables  occupy  more  of  a  position  in  the  menu 
than  is  too  often  the  case. 

There  has  recently  appeared  a  crusade  for  the  eating  of  yeast 
cakes.  The  claim  made  for  their  use  rests  on  a  perfectly  firm 
basis,  they  are  rich  in  the  "B"  vitamine,  the  proteins  of  the 
yeast  cake  are  of  good  quality  and  the  cake  contains  no  ingre- 
dients poisonous  to  man.  Many  people  are  reporting  beneficial 
effects  from  their  use.  Is  there  any  lesson  to  be  drawn  from 
this  experiment?  I  feel  that  the  very  fact  that  benefits  have 
resulted  from  this  yeast  feeding  is  excellent  evidence  of  lack  of 
the  vitamine  in  the  diets  of  the  people  affected  and  a  clear  argu- 
ment that  the  dietary  habits  of  many  people  need  adjustment  to 


86  VITAMINE   MANUAL 

a  higher  vitamine  content.  Whether  it  is  necessary  to  use  yeast 
cakes  or  any  other  concentrate  of  vitamine,  depends  entirely 
upon  whether  the  ordinary  diet  is  lacking  in  these  factors  and  my 
first  advice  in  the  matter  would  be  to  make  if  possible  a  selection 
of  the  vitamine  containing  foods  and  see  if  normal  conditions  did 
not  result  before  utilizing  foods  whose  taste  is  not  pleasing  or 
which  are  taken  as  medicine.  For  it  is  an  old  experience  that 
medicines  will  be  taken  only  so  long  as  the  patient  is  sick  and 
perhaps  it  is  just  as  well  so.  In  other  words  I  believe  it  is  pos- 
sible with  intelligent  selection  based  on  such  tables  as  are  given 
in  Chapter  IV  for  people  to  secure  from  the  butcher  and  the 
grocer  all  their  requirements  of  these  vitamines  as  a  part  of  their 
regular  palatable  diet.  To  those  who  have  neglected  this  selec- 
tion and  find  remedy  in  concentrates,  that  fact  should  lead  them 
to  reconstruct  their  diet  rather  than  persist  in  dependence  on  the 
medicine  to  correct  faulty  diet.  In  other  words  the  same  argu- 
ments apply  to  the  use  of  medicinal  concentrates  of  vitamines 
as  applies  to  the  use  of  laxatives.  At  times  these  substances  are 
very  valuable  as  cures,  but  it  is  better  by  far  to  so  regulate  the 
dietary  habits  as  to  avoid  the  necessity  for  their  use. 

Another  phase  of  this  matter  that  promises  to  develop  in  the 
near  future  as  a  result  of  the  vitamine  hypothesis  is  a  reform  in 
food  manufacture.  There  has  been  a  strong  tendency  during  the 
past  two  decades  to  "purify"  food  products.  The  genesis  of  this 
tendency  is  to  be  found  in  a  highly  laudable  ambition  to  force  the 
manufacturer  to  eliminate  impurities  and  adulterations  and 
provide  clean,  wholesome,  sanitary  food.  Unfortunately  in 
attempting  to  meet  this  demand  on  the  part  of  the  public,  the 
food  manufacturer  has  sometimes  neglected  to  seek  advice  from 
the  nutrition  expert  and  the  latter  has  failed  to  appreciate  the 
need  of  advice.  The  net  result  has  been  to  discover  that  Nature 
is  often  a  better  chemist  than  man  and  has  a  much  better  knowl- 
edge of  what  man  needs  in  his  diet  than  the  chemist.  The 
chemist  employed  by  the  manufacturer  has,  as  a  result,  gone  to 
such  a  limit  in  his  development  of  purification  methods  as  to 
often  eliminate  the  essential  nutrients  and  the  result  has  been 
foods  that  will  stand  analysis  for  pure  nutrients,  but  which  will 
not  stand  Nature's  analysis  for  dietary  efficiency.  As  a  second- 


THE   VITAMINES   IN   DIETS  87 

ary  result  of  this  tendency  we  have  acquired  habits  that  in  many 
cases  must  either  be  broken  or  must  have  grafted  on  to  them 
other  habits  which  shall  remedy  the  defective  ones.  Take  the 
milling  of  wheat  as  an  example.  Nature  put  into  the  wheat  grain 
most  of  the  elements  needed  by  man  and  in  the  early  days  he  was 
content  to  grind  up  the  whole  grain  and  find  it  palatable.  The 
craze  for  purity  as  expressed  by  color  has  gradually  replaced  this 
whole  meal  wheat  with  a  beautiful  white  product  that  is  largely 
pure  starch  with  a  few  of  the  proteins  retained.  And  the  princi- 
pal protein  retained  lacks  one  of  the  greatest  essentials  for  growth 
while  the  vitamines  have  all  been  practically  eliminated  with  the 
grain  germ.  Intelligence  tells  us  then  that  if,  having  formed  the 
habit,  we  will  persist  in  our  appetite  for  white  flour  we  must  see 
to  it  that  the  protein  deficiency  of  the  latter  and  its  lack  of  vita- 
mines  is  compensated  for  by  supplementing  the  diet  with  the 
food-stuffs  in  which  these  are  rich.  We  may  in  other  words 
retain  our  bad  habits  in  taste  if  we  will  graft  on  to  them  the 
attention  to  the  eliminated  factors  and  their  substitution  in  other 
form. 

In  general  then,  the  adult  needs  to  review  his  feeding  habits 
and  analyze  them  in  the  light  of  our  new  knowledge.  For  this 
purpose  the  tables  of  Chapter  IV  supply  data  useful  so  far  as 
vitamines  are  concerned,  but  it  will  be  perhaps  worth  while  to 
repeat  here  some  of  this  data  in  more  generalized  form. 

a.  Sources  of  the  "A"  vitamine 

Its  most  abundant  sources  are  milk,  butter,  egg  yolk  fat,  and 
the  green  leaves  of  plants  usually  classed  as  salads.  Cabbage, 
lettuce,  spinach  and  carrots  contain  this  substance  in  considerable 
quantity.  The  germ  of  cereals  is  fairly  rich  in  the  factor,  but  the 
rest  of  the  grain  is  deficient  and  white  flours  are  therefore  poorer 
than  whole  meals  in  this  respect.  Cooking  temperatures  have 
little  effect  on  this  vitamine  and  hence  little  attention  need  be 
paid  to  cooking  temperatures  as  far  as  this  vitamine  is  concerned. 


88  VITAMINE   MANUAL 

b.  Sources  of  the  "B"  vitamine 

Its  principal  sources  outside  of  yeast  are  the  seeds  of  plants 
and  the  eggs  and  milk  of  animals.  Meat  contains  relatively 
little  of  this  substance  but  glandular  organs  such  as  the  liver 
and  pancreas  are  fairly  rich  in  it.  In  the  seeds  the  distribution 
is  general  throughout  the  whole  body  of  the  seed  in  the  case  of 
beans,  peas,  etc.,  but  in  the  cereal  grains  it  is  largely  restricted  to 
the  embryo  portion  and  hence  a  high  degree  of  milling  tends  to 
reduce  the  per  cent  of  this  factor  in  any  highly  milled  cereal. 
White  flour  and  polished  rice  are  notable  examples  of  deficiency 
of  "B"  vitamine  due  to  this  milling  process.  Fruits  such  as 
oranges,  tomatoes,  and  lemons  are  good  sources  and  there  is  a 
fair  amount  present  in  the  apples  and  grapes  and  other  common 
food  fruits.  Many  vegetables  show  it  in  fair  abundance,  notably 
potatoes,  carrots,  and  turnips,  but  the  rule  is  not  general  for 
beets  are  extremely  poor  in  this  factor.  Nuts  are  also  good 
sources.  Eggs,  milk  and  cheese  contain  it  in  fair  abundance. 
Cooking  temperatures  have  little  effect  on  this  type  if  the  temper- 
ature does  not  clirnb  above  the  boiling  point  and  if  the  cooking 
water  is  not  "  alkaline."  In  the  latter  case  it  becomes  necessary 
to  determine  the  extent  of  destruction  and  either  eat  enough  to 
insure  protection,  or  reform  the  method  of  cookery. 

c.  Sources  of  the  "C"  vitamine 

Its  richest  sources  are  vegetables  such  as  cabbage,  swedes, 
turnips,  lettuce  and  watercress;  fruits  such  as  lemons,  oranges, 
raspberries  and  tomatoes.  Certain  of  the  vegetables  such  as 
potatoes  have  a  substantial  value  in  this  respect,  but  meat  and 
most  prepared  milks  are  low  in  antiscorbutic  values.  The  sus- 
ceptibility of  this  vitamine  to  drying,  heat  and  alkali,  make  it 
necessary  to  scrutinize  your  cooking  methods  very  carefully  in 
order  not  to  ruin  a  good  source  by  a  poor  preparation  of  it  for 
the  table. 


CHAPTER  VIII 

AVITAMINOSES  OR  THE  DISEASES  THAT  RESULT  FROM  VlTAMINE 

DEFICIENCIES 

A  survey  of  the  vitamines  would  be  incomplete  without  a  dis- 
cussion of  the  vitamine  deficiency  diseases  in  particular,  though 
many  of  the  facts  already  cited  obviously  bear  on  the  treatment 
and  prevention  of  such  diseases. 

The  idea  of  "avitaminoses"  or  vitamine  deficiency  as  the 
cause  of  a  disease  of  a  specific  nature  was  set  forth  in  detail  by 
Funk  in  his  book  Die  Vitamine.  In  his  discussion  of  this  view 
he  suggests  several  types  that  would,  he  felt,  on  examination 
prove  to  be  due  to  the  absence  of  a  vitamine  in  the  diet.  Of 
these  predicted  types  beri-beri  was  the  only  one  to  be  established 
in  1913.  Scurvy  has  now  been  added  to  the  fold  and  rickets  or 
rachitis  seems  well  on  the  way  to  acceptance  though  the  specific 
vitamine  absent  in  this  case  is  not  yet  positively  identified. 
Pellagra  still  resists  the  efforts  of  the  vitamine  hypothesis  to  bend 
it  to  that  theory  and  its  etiology  is  still  obscure. 

I.    BERI-BERI 

This  disease  while  specifically  confined  to  the  oriental  in  the 
mind  of  the  student  can  be  justly  considered  of  much  wider  dis- 
tribution for  the  mild  forms  of  malnutrition  associated  with  a 
deficiency  in  the  "B"  vitamine  are  less  acute  manifestations  of 
this  disease.  The  disease  is  not  likely  to  become  marked  in  well 
nourished  districts  in  its  acute  form,  but  in  famine  districts  its 
incidence  is  always  possible.  It  would  be  more  than  possible 
were  it  not  for  the  fact  that  famine  tends  to  eliminate  the  highly 
milled  cereals  and  throw  the  people  back  on  to  the  whole  grain, 
peas  and  beans,  which  are  rich  in  the  preventive  factor.  But 
when  for  any  reason  diets  become  limited  extra  attention  is 
demanded  in  regard  to  their  selection  and  preparation.  The 
main  characteristics  of  this  disease  have  already  been  fully  cov- 
ered in  what  precedes  and  need  not  be  repeated  here. 


90  VITAMIN  E   MANUAL 

II.   SCUEVY 

This  disease,  like  beri-beri  has  already  been  fully  discussed  in 
what  precedes.     One  of  the  striking  discoveries  of  this  subject 
has  been  the  retreat  from  favor  of  the  time-honored  lime  juice 
which  is  now  found  to  be  much  less  potent  than  oranges,  lemons, 
or  even  canned  tomato  juice  and  which  on  preservation  loses 
practically  all  its  potency.     In  the  modern  hospital,  cases  of 
scurvy  rarely  appear  ouside  of  occasional  infant  cases  and  it 
might  appear  that  the  problem  of  scurvy  prevention  is  peculiarly 
that  of  the  sailor,  the  explorer  and  the  army  rationer.     Never- 
theless an  insufficient  supply  of  the  "C"  vitamine  may  retard 
growth  and  well  being  in  the  individual  without  manifesting  itself 
in  its  more  acute  form  of  scurvy.     In  a  recent  review  Hess  states: 
"It  is  hardly  an  exaggeration  to  state  that  in  the  temperate  zones 
the  development  or  non-development  of  scurvy  depends  largely 
on  the  potato  crop."     "This  is  attributed  in  part  to  the  fact  that 
the  potato  is  an  excellent  antiscorbutic,  but  to  a  greater  extent 
because  it  is  consumed  during  the  winter  in  amounts  that  exceed 
the  combined  total  of  all  other  vegetables."    To  the  public  and 
to  the  food  purveyor  there  is  a  definite  problem  in  how  to  best 
supply  the  preventive  and  how  best  to  concentrate  and  preserve 
the  sources  of  this  vitamine  without  injury  to  its  potency.     The 
following  observation  is  therefore  appended  as  bearing  on  this 
point.     In  the  absence  of  fruits  or  other  high  potency  sources  it 
is  possible  to  develop  this  factor  in  cereal  grains  by  the  simple 
expediency  of  sprouting.    If  seeds  are  soaked  in  water  for  twenty- 
four  hours  and  then  kept  moist  for  from  one  to  three  days  with 
the  free  access  of  air,  sprouts '  will  develop  whose  content  of  the 
antiscorbutic  vitamine  is  comparable  to  that  of  many  fresh  vege- 
tables, even  though  the  dry  seeds  themselves  have  little  of  this 
factor.     In  other  words  the  germination  process  is  a  synthesiser 
of  the  vitamine.     This  observation  may  be  of  value  where  fruits 
and  vegetables  are  scarce  or  expensive.     On  account  of  cooking 
effects,  it  cannot  be  too  often  reiterated  that  raw  fruits,  vege- 
tables and  salads,  are  of  more  value  than  cooked  forms  of  these 
same  sources  and  that  drying  processes  are  extremely  destructive 
where  heat  enters  into  the  drying  process.    Vacuum  drying  seems 
to  be  much  less  destructive  and  it  may  be  possible  to  develop  the 


AVITAMINOSES  91 

drying  of  vegetables  to  a  point  where  retention  of  this  vitamine 
factor  is  practical.  At  present  all  dried  vegetables  should  be 
regarded  with  suspicion  as  a  source  of  vitamine  "C."  Expressed 
juices  may  often  be  used  where  the  whole  vegetable  is  scarce 
or  incompatible  and  this  fact  is  one  to  be  borne  in  mind  by  the 
worker  in  famine  districts. 

in.  RACHITIS   (RICKETS) 

This  disease  is  engaging  the  attention  of  many  workers  on  both 
sides  of  the  Atlantic  at  the  present  time.  In  England  the  princi- 
pal contributor  is  Dr.  Mellanby,  who  has  accumulated  evidence 
which  he  believes  indicates  that  the  preventive  factor  is  the  A 
vitamine.  This  view  is  not  yet  accepted  as  conclusive  by  the 
American  workers.  McCollum,  Rowland,  Park,  and  others  at 
Johns  Hopkins  University  have  experimented  with  various  rickets- 
producing  diets  and  while  the  principal  deficiency  in  these  diets 
seems  to  be  Ca  salts  and  the  A  vitamine  they  do  not  consider 
that  the  disease  can  as  yet  be  traced  to  deficiency  in  any  one 
factor.  Hess  has  called  attention  to  several  new  features  and  the 
significance  of  some  older  measures.  He  has  shown  on  the  one 
hand  that  cod-liver  oil  is  almost  a  specific  remedy  for  the  disease 
but  that  this  remedy  is  not  replaceable  by  other  rich  sources  of 
the  A  vitamine.  He  has  also  recently  shown  that  hygienic  meas- 
ures may  have  an  influence.  Schmorl  showed  that  the  disease  was 
seasonal,  a  high  rate  maintaining  in  the  winter  months  and  a 
lower  rate  in  the  summer  months.  Hess  has  recently  reported 
beneficial  results  from  use  of  the  ultra-violet  rays  which  he  uses 
as  a  substitute  for  sunlight.  The  results  seem  to  confirm 
SchmorPs  view  that  the  sunlight  of  the  summer  months  is  a 
preventive  factor.  He  has  also  suggested  that  the  specific  effect 
of  the  cod-liver  oil  might  be  due  to  a  new  vitamine,  Vitamine  D? 
On  the  other  hand  Zilva  and  Miura  in  England  have  recently 
shown  that  crude  cod-liver  oil  is  something  like  two  hundred  and 
fifty  times  as  rich  in  vitamine  A  as  butter  fat,  which  tends  to 
support  the  British  view  that  the  A  vitamine  is  the  antirachitic 
factor. 

Sherman  and  Pappenheimer  have  recently  shown  that  the 
phosphates  exert  a  marked  preventive  effect  on  rickets  and  sug- 


92  VITAMINS   MANUAL 

gest  that  the  utilization  of  the  calcium  by  the  individual  may  be 
determined  in  part  by  this  factor. 

The  views  in  brief  are  now  in  an  extremely  chaotic  state  and 
it  is  impossible  at  present  to  determine  whether  rickets  is  a  true 
avitaminose  or  a  consequence  of  deficiency  in  a  series  of  factors. 
It  is  however  certain  that  the  disease  in  its  subacute  forms  is 
extremely  wide-spread  among  infants  and  that  its  prevention 
can  be  most  easily  secured  by  the  addition  of  cod-liver  oil  to  the 
diet.  In  this  procedure  warning  is  necessary  that  the  cod-liver 
oil  be  as  pure  a  product  of  oil  as  possible,  since  the  market  prepara- 
tions are  often  almost  devoid  of  the  true  oil  and  hence  of  the 
curative  agent. 

IV.   PELLAGBA 

This  disease  has  been  the  subject  of  exhaustive  inquiry  and 
study  on  this  side  of  the  Atlantic  and  the  findings  of  the  various 
investigating  boards  have  added  much  to  the  prevention  and 
cure  of  the  scourge,  but  have  failed  as  yet  to  agree  on  any  one 
etiological  factor.  The  best  recent  review  of  the  current  findings 
is  to  be  found  in  an  article  by  Voegtlin  published  as  Reprint  597 
of  the  Public  Health  Reports  of  the  United  States  Public  Health 
Service.  His  conclusions  may  be  quoted  in  full  as  representing 
the  latest  summary  of  evidence  now  extant: 

1.  The  hypothesis  that  there  is  a  causal  relation  between  pellagra  and 
a  restricted  vegetable  diet  has  been  substantiated  by  direct  proof  to  this 
effect  and  has  led  to  results  of  considerable  practical  and  scientific  value. 

2.  The  metabolism  in  pellagra  shows  certain  definite  changes  from  the 
normal,  which  point  to  decreased  gastric  secretion  and  increased  intestinal 
putrefaction. 

3.  In  the  treatment  and  prevention  of  pellagra,  diet  is  the  essential 
factor.    The  disease  can  be  prevented  by  an  appropriate  change  in  diet 
without  changing  other  sanitary  conditions. 

4.  A  diet  of  the  composition  used  by  pellagrins  prior  to  their  attack  by 
the  disease  leads  to  malnutrition  and  certain  pathological  changes  in 
animals,  resembling  those  found  in  pellagra.    A  typical  pellagrous  derma- 
titis has  not  been  observed  in  animals.    Pellagrous  symptoms  have  been 
produced  in  man  by  the  continued  consumption  of  a  restricted  vegetable 
diet. 

5.  The  nature  of  the  dietary  effect  has  not  been  discovered,  although  certain 
observations  point  to  a  combined  deficiency  in  some  of  the  recognized  diet- 
ary factors  as  the  cause  of  the  pellagrous  syndrome. 


AVITAMINOSES  93 

In  elaborating  on  conclusion  5  Voegtlin  states  that: 

The  conception  that  pellagra  is  due  to  a  dietary  deficiency  is,  therefore, 
not  contradicted  by  the  available  evidence.  This  does  not  imply  that  the 
disease  is  necessarily  due  to  a  deficiency  of  diet  in  a  specific  substance  sueh 
as  the  hypothetical  pellagra  vitamine  of  Funk  (1913).  It  is  much  more 
likely  that  the  pellagrous  syndrome  is  caused  by  a  combination  of  the 
deficiencies  in  some  of  the  well  recognized  food  factors. 

V.   OTHER    AVITAMINOSES 

The  r61e  of  the  vitamine  in  the  nutrition  and  growth  of  organ- 
isms other  than  the  man  is  becoming  a  matter  of  interest  in  vari- 
ous ways.  The  construction  of  culture  media  for  various  strains 
of  bacteria  and  the  conditions  favorable  or  unfavorable  to  their 
growth,  are  features  of  study  in  which  the  new  hypothesis  has 
demanded  attention.  It  has  already  been  claimed  that  vitamines 
are  essential  to  the  growth  of  the  meningococcus,  the  influenza 
bacillus,  the  typhoid  bacillus,  the  gonococcus,  the  pneumococcus 
Type  I,  Streptococcus  hemolyticus,  the  diptheria  bacillus,  the 
Bacillus  pertussis  and  certain  soil  organisms.  If  these  views  are 
confirmed  it  becomes  evident  that  the  means  for  prevention  of 
the  development  of  these  forms  may  lie  in  the  control  of  the 
vitamine  content  of  the  materials  on  which  these  forms  thrive  and 
that  in  the  study  of  these  types  it  may  be  possible  to  speed  up 
the  incubation  of  strains  and  thus  hasten  diagnostic 'measures  by 
introducing  the  necessary  vitamines  into  the  culture  media. 
These  observations  merely  suggest  the  possible  widening  of  the 
scope  of  the  vitamine  study  in  the  service  of  man  and  give  added 
reason  for  our  keeping  pace  with  the  strides  made  in  this  par- 
ticular field. 


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Med.  Journ.,  1920,  i,  249. 

MCCARRISON,  R. :  The  genesis  of  edema  in  beri-beri.  Proc.  Roy.  Soc.,  1920, 
B,  91  &  103. 


110  VITAMINE   MANUAL 

McCARRisoN,  R. :  The  pathogenesis  of  deficiency  disease.  Indian  Journ. 
Med.  Research,  1919,  vi,  275,  550;  vii,  167,  188,  269,  279,  308, 
342,  633. 

MCCLENDON,  J.  F.,  COLE,  W.  C.  C.,  ENGSTRAND,  O.,  AND  MIDDLEKAUFF, 
J.  E. :  The  effect  of  Malt  and  malt  extracts  on  scurvy  and  on 
the  alkaline  reserve  of  the  blood.  J.  Biol.  Chem.,  1919,  xl,  243. 

MCCLENDON,  J.  F.,  AND  SHARP,  P.  F. :  Determination  of  the  pH  concen- 
tration of  foods  during  storage  and  preparation  in  relation  to 
the  preservation  of  antiscorbutic  properties.  J.  Biol.  Chem., 
1920,  xli,  p.  iv  Proc. 

MCCLENDON,  J.  F.:  Nutrition  and  public  health  with  special  reference 
to  vitamines.  Amer.  J.  Med.  Sciences,  1920,  clix,  477. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M.  i  The  necessity  of  certain  lipins  in  the 
diet  during  growth.  J.  Biol.  Chem.,  1913,  xv,  167. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M. :  Proc.  Soc.  Exp.  Biol.  and  Med.,  1913-14, 
xi,  101. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M. :  Observations  on  the  isolation  of  the 
substance  in  butter  fat  that  exerts  a  stimulatory  action  upon 
growth.  J.  Biol.  Chem.,  1914,  xix,  245. 

MCCOLLTJM,  E.  V. :  The  value  of  the  proteins  of  the  cereal  grains  and  milk 
for  growth  in  the  pig  and  the  influence  of  the  plane  of  protein 
intake  on  growth.  J.  Biol.  Chem.,  1914,  xix,  323. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M.  I  The  influence  of  the  plane  of  protein 
intake  in  growth.  J.  Biol.  Chem.,  1915,  xx,  415. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M. :  Nutrition  with  purified  food  substances. 
J.  Biol.  Chem.,  1915,  xx,  641. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M.  i  The  influence  of  certain  vegetable  fats 
on  growth.  J.  Biol.  Chem.,  1915,  xxi,  179. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M. :  The  influence  of  the  composition  and 
amount  of  mineral  content  of  the  ration  on  growth  and  repro- 
duction. J.  Biol.  Chem.,  1915,  xxi,  615. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M.  I  The  nature  of  the  dietary  deficiencies 
of  rice.  J.  Biol.  Chem.,  1915,  xxiii,  181. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M.  i  The  essential  factors  in  the  diet  during 
growth.  J.  Biol.  Chem.,  1915,  xxiii,  231. 

McCoLLUM,  E.  V.,  AND  DAVIS,  M. :  The  cause  of  the  loss  of  the  nutritive 
efficiency  of  heated  milk.  J.  Biol.  Chem.,  1915,  xxiii,  247. 

McCoLLUM,  E.  V.,  SIMMONDS,  N.,  AND  PiTz:  Proc.  Soc.  Exp.  Biol.  and 
Med.,  1916,  xiii,  129. 

McCoLLUM,  E.  V.,  AND  KENNEDY:  Dietary  factors  operating  in  the  pro- 
duction of  polyneuritis.  J.  Biol.  Chem.,  1916,  xxiv,  491. 

McCoLLUM,  SIMMONDS  AND  PITZ  :  The  relation  of  the  unidentified  dietary 
factors  fat-soluble  A  and  water-soluble  B  of  the  diet  to  the 
growth  promoting  properties  of  milk.  J.  Biol.  Chem.,  1916, 
xxvii,  33. 

McCoLLUM,  SIMMONDS  AND  PITZ:  The  nature  of  the  dietary  deficiencies 
of  the  wheat  embryo.  J.  Biol.  Chem.,  1916,  xxv,  105. 


BIBLIOGRAPHY  111 

McCoLLUM,  SIMMONDS  AND  PITZ  :  The  vegetarian  diet  in  the  light  of  our 
present  knowledge  and  the  distribution  in  plants  of  fat-soluble 
A.  Am.  J.  Physiol.,  1916,  xli,  333,  361  and  374. 

McCoLLUM,  SIMMONDS  AND  PITZ:  The  dietary  deficiencies  of  the  maize 
kernel.  J.  Biol.  Chem.,  1916-17,  xxviii,  153. 

McCoLLUM,  SIMMONDS  AND  PITZ  :  The  effect  of  feeding  the  proteins  of  the 
wheat  kernel  at  different  planes  of  intake.  J.  Biol.  Chem., 
1916-17,  xxviii,  211. 

McCoLLUM,  SIMMONDS  AND  PITZ  :  Is  lysine  the  limiting  amino  acid  in  pro- 
teins of  wheat,  maize  and  oats?  J.  Biol.  Chem.,  1916-17,  xxviii, 
483. 

MCCOLLUM,  E.  V. :  The  Harvey  Lectures,  1916-17,  xii,  151. 

McCoLLUM,  SIMMONDS  AND  PITZ:  The  nature  of  the  dietary  deficiencies 
of  the  oat  kernel.  J.  Biol.  Chem.,  1917,  xxix,  341. 

McCoLLUM,  SIMMONDS  AND  PITZ:  The  dietary  deficiencies  of  the  white 
bean.  J.  Biol.  Chem.,  1917,  xxix,  521. 

McCoLLUM,  SIMMONDS  AND  PITZ:  The  supplementary  dietary  relations 
between  leaf  and  seed  as  contrasted  with  combinations  of  seed 
with  seed.  J.  Biol.  Chem.,  1917,  xxx,  13. 

McCoLLUM  AND  PITZ:  The  vitamine  hypothesis  and  deficiency  diseases. 
J.  Biol.  Chem.,  1917,  xxxi,  229. 

McCoLLUM,  E.  V.,  AND  SIMMONDS  :  A  biological  analysis  of  pellagra  pro- 
ducing diets.  I.  The  dietary  properties  of  mixtures  of  maize 
kernel  and  bran.  J.  Biol.  Chem.,  1917,  xxxii,  29. 

McCoLLUM,  E.  V.,  AND  SIMMONDS  :  A  biological  analysis  of  pellagra  pro- 
ducing diets.  II.  The  minimum  requirements  of  the  two  un- 
identified dietary  factors  for  maintenance  as  contrasted  with 
growth.  J.  Biol.  Chem.,  1917,  xxxii,  347. 

McCoLLUM,  E.  V.,  AND  SIMMONDS  :  A  biological  analysis  of  pellagra  pro- 
ducing diets.  III.  The  values  of  some  seed  proteins  for  main- 
tenance. J.  Biol.  Chem.,  1917,  xxxii,  347. 

McCoLLUM,  E.  V. :  The  supplementary  dietary  relations  among  our  nat- 
ural food  stuffs.  J.  Am.  Med.  Assn.,  1917,  Ixviii,  1379. 

McCoLLUM  AND  SIMMONDS  :  A  study  of  the  dietary  essential,  water-soluble 
B  in  relation  to  its  solubility  and  stability  toward  reagents. 
J.  Biol.  Chem.,  1918,  xxxiii,  55. 

McCoLLUM  AND  SIMMONS  :  Pellagra  IV.  The  causes  of  failures  of  mixtures 
of  seeds  to  promote  growth  in  young  animals.  J.  Biol.  Chem., 
1918,  xxxiii,  303. 

McCoLLUM  AND  SIMMONS  :  Pellaga  V.  The  nature  of  the  dietary  defi- 
ciencies of  a  diet  derived  from  peas,  wheat  flour  and  cotton  seed 
oil.  J.  Biol.  Chem.,  1918,  xxxiii,  411. 

McCoLLUM,  SIMMONDS  AND  PARSONS  :  The  dietary  properties  of  the  potato. 
J.  Biol.  Chem.,  1918,  xxxvi,  197. 

McCoLLUM,  E.  V.,  AND  SIMMONDS  :  The  nursing  mother  as  a  factor  of  safety 
in  the  nutrition  of  the  young.  Am.  J.  Physiol.,  1918,  xlvi,  275. 


112  VITAMINE   MANUAL 

McCoLLUM,  E.  V. :  The  Newer  Knowledge  of  Nutrition,  a  book.  Mac- 
millan,  1919. 

McCoLLUM,  SIMMONDS  AND  PARSONS:  The  dietary  properties  of  the  pea. 
J.  Biol.  Chem.,  1919,  xxxvii,  287 

McCoLLUM  AND  PARSONS:  The  etiology  of  rickets.  J.  Biol.  Chem.,  1920, 
xli,  p.  xxxi  of  the  proceedings. 

McCoLLUM  AND  PARSONS  i  The  antiscorbutic  requirements  of  the  Prairie 
dog.  J.  Biol.  Chem.,  1920,  xliv,  609. 

McCoLLUM,  SIMMONDS,  SHIPLEY,  AND  PARK.  :  Studies  on  the  experimental 
production  of  rickets.  I.  The  production  of  rickets  and  similar 
diseases  in  the  rat  by  different  diets.  J.  Biol.  Chem.,  1921,  xlv, 
333;  and  II.  The  effect  of  cod-liver  oil  administered  to  rats  with 
experimental  rickets.  J.  Biol.  Chem.,  1921,  xlv,  343 

MAcDouGAL,  D.  J. :  The  effects  of  yeast  vitamine  water-soluble  B  on 
plant  cell  masses  and  on  biocolloids.  Proc.  Soc.  Exp.  Biol.  and 
Med. ,  1920,  xviii,  39. 

MACDONALD  AND  McCoLLUMi  J.  Biol.  Chem.,  1921,  xlv,  307. 

MACKAY,  H.  M.  M. :  Observations  on  rickets  in  an  out-patient  department. 
Brit.  Med.  Journal,  1920,  II,  929. 

MACKENZIE-WALLIS,  R.  L. :  Food  value  of  the  ground  nut  (Arachis).  Ind. 
J.  Med.  Res.,  1918,  vi,  45. 

NELSON,  V.  E.,  AND  LAMB,  A.  R. :  Vitamine  deficiency.  I.  Xerophthalmia 
in  rickets.  Am.  J.  Physiol.,  1920,  li,  530. 

NELSON,  V.  E.,  FULMER,  E.  L.,  AND  CESSNA,  R.:  The  nutritional  require- 
ments of  yeast.  III.  The  synthesis  of  the  water-soluble  B  by 
the  yeast.  J.  Biol.  Chem.,  1921,  xlvi,  77. 

NESBITT,  CHAS.  T. :  Sanitation  in  the  control  of  pellagra.  J.  Am.  Med. 
Assn.,  1916,  Ixvi,  647. 

NORTHROP:  The  r61e  of  yeast  in  the  nutrition  of  an  insect  (Drosophila). 
J.  Biol.  Chem.,  1917,  xxx,  181. 

NOVARO,  P. :  Gaz.  degli,  Ospedali,  Milan,  1920,  xli,  424. 

NOVARO,  P.:  Richerche  calorimetriche  comparative  sul  diguino  e  sull 
avitaminosi.  Pathologica,  1920,  xii,  133. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Feeding  experiments  with  isolated 
food  substances.  Pub.  Carnegie  Inst.  Bull.,  1911,  Pts.  I  and 
II,  No.  156. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Feeding  experiments  with  fat-free 
food  mixtures.  J.  Biol.  Chem.,  1912,  xii,  81. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  role  of  gliadin  in  nutrition.  J. 
Biol.  Chem.,  1912,  xii,  473. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Maintenance  experiments  with  iso- 
lated proteins.  J.  Biol.  Chem.,  1913,  xiii,  233. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  relation  of  growth  to  the  chemi- 
cal constituents  of  the  diet.  J.  Biol.  Chem.,  1913,  xv,  311. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  influence  of  butter  fat  on  growth. 
J.  Biol.  Chem.,  1913-14,  xvi,  423. 


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OSBORNE,  T.  B.,  AND  MENDEL,  L.  B.  i  Amino  acids  in  nutrition.  J.  Biol. 
Chem.,  1914,  xvii,  325. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  influence  of  cod -liver  oil  and 
some  other  fats  on  growth.  J.  Biol.  Chem.,  1914,  xvii,  401. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Nutritive  properties  of  the  maize 
kernel.  J.  Biol.  Chem.,  1914,  xviii,  1. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B.:  The  repression  of  growth  and  the 
capacity  to  grow.  J.  Biol.  Chem.,  1914,  xviii,  95. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Further  observations  on  the  influence 
of  natural  fats  on  growth.  J.  Biol.  Chem.,  1915,  xx,  379. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B.:  The  comparative  nutritive  value  of 
certain  proteins  and  the  problem  of  protein  minimum.  J.  Biol. 
Chem.,  1915,  xx,  351. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Protein  minima  for  maintenance. 
J.  Biol.  Chem.,  1915,  xxii,  241. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  resumption  of  growth  after  long- 
continued  failure  to  grow.  J.  Biol.  Chem.,  1915,  xxiii,  439. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  stability  of  the  growth-promot- 
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OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  amono  acid  minimum  for  main- 
tenance and  growth  as  exemplified  by  further  experiments  with 
lysine  and  tryptophane.  J.  Biol.  Chem.,  1916,  xxv,  1. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  A  quantitative  comparison  of  casein, 
lactalbumin  and  edestin  for  growth  and  maintenance.  J.  Biol. 
Chem.,  1916,  xxvi,  1. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  effect  of  the  amino  acid  content 
of  the  diet  upon  the  growth  of  chickens.  J.  Biol.  Chem.,  1916, 
xxvi,  293. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  relative  value  of  certain  proteins 
and  protein  concentrates  as  supplements  to  corn  gluten.  J. 
Biol.  Chem.,  1917,  xxix,  69. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  use  of  cotton-seed  as  food.  J. 
Biol.  Chem.,  1917,  xxix,  289. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  role  of  vitamines  in  the  diet. 
J.  Biol.  Chem.,  1917,  xxxi,  149. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B.:  Nutritive  factors  in  animal  tissues. 
J.  Biol.  Chem.,  1917,  xxxii,  309. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  The  use  of  the  soy-bean  as  food.  J. 
Biol.  Chem.,  1917,  xxxii,  369. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Some  new  constituents  of  milk.  J. 
Biol.  Chem.,  1918,  xxxiii,  243. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B.:  The  growth  of  chickens  in  confine- 
ment. J.  Biol.  Chem.,  1918,  xxxiii,  433. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Nutritive  factors  in  animal  tissues. 
II.  J.  Biol.  Chem.,  1918,  xxxiv,  17. 

OSBORNE  AND  MENDEL:  The  inorganic  elements  in  nutrition.  J.  Biol. 
Chem.,  1918,  xxxiv,  131. 


114  VITAMINE   MANUAL 

OSBORNE  AND  MENDEL:  Nutritive  factors  in  plant  tissues.  I.  The  protein 
factors  in  the  seeds  of  cereals.  J.  Biol.  Chem.,  1918,  xxxiv,  521. 

OSBORNE  AND  MENDEL:  Milk  as  a  source  of  water-soluble  vitamin.  J. 
Biol.  Chem.,  1918,  xxxiv,  537. 

OSBORNE  AND  MENDEL:  The  choice  between  an  adequate  and  inadequate 
diet  as  made  by  rats.  J.  Biol.  Chem.,  1918,  xxxv,  19. 

OSBORNE  AND  MENDEL:  The  vitamines  in  green  foods.    J.  Biol.  Chem., 

1919,  xxxvii,  187. 

OSBORNE  AND  MENDEL:  A  method  of  expressing  numerically  the  growth- 
promoting  value  of  proteins.  J.  Biol.  Chem.,  1919,  xxxvii,  223. 

OSBORNE  AND  MENDEL:  The  nutritive  value  of  the  wheat  kernel  and  its 
milling  products.  J.  Biol.  Chem.,  1919,  xxxvii,  557. 

OSBORNE  AND  MENDEL:  The  nutritive  value  of  yeast  protein.  J.  Biol. 
Chem.,  1919,  xxxviii,  223. 

OSBORNE  AND  MENDEL:  Nutritive  factors  in  plant  tissues.  II.  The  dis- 
tribution of  water-soluble  vitamin.  J.  Biol.  Chem.,  1919,  xxxix, 
29. 

OSBORNE  AND  MENDEL:  Nutritive  value  of  the  proteins  of  the  barley,  oat, 
rye  and  wheat  kernel.  J.  Biol.  Chem.,  1920,  xli,  275. 

OSBORNE  AND  MENDEL:  Nutritive  factors  in  plant  tissues.  III.  Further 
observations  on  the  distribution  of  water-soluble  vitamin.  J. 
Biol.  Chem.,  1920,  xli,  451. 

OSBORNE  AND  MENDEL:  Fat  soluble  vitamine  of  green  foods.  J.  Biol. 
Chem.,  1920,  xli,  p.  vii. 

OSBORNE  AND  MENDEL:  Milk  as  a  source  of  water  soluble  vitamin  II.  J. 
Biol.  Chem.,  1920,  xli,  515. 

OSBORNE  AND  MENDEL:  Nutritive  factors  in  plant  tissues.  IV.  Fat-soluble 
vitamine.  J.  Biol.  Chem.,  1920,  xli,  549. 

OSBORNE  AND  MENDEL:  The  occurrence  of  water-soluble  vitamin  in  some 
common  fruits.  J.  Biol.  Chem.,  1920,  xlii,  465. 

OSBORNE  AND  MENDEL  :  Skimmed  milk  as  a  supplement  to  corn  in  feeding. 
J.  Biol.  Chem.,  1920,  xliv,  1. 

OSBORNE  AND  MENDEL:  A  critique  of  experiments  with  diets  free  from 
fat-soluble  vitamin.  J.  Biol.  Chem.,  1921,  xlv,  277. 

OSBORNE  AND  MENDEL:  Growth  on  diets  poor  in  true  fats.    J.  Biol.  Chem., 

1920,  xlv,  145. 

OSBORNE  AND  MENDEL:  Do  fruits  contain  water-soluble  B.  Proc.  Soc. 
Exper.  Biol.  and  Med.,  1919,  xvii,  46. 

OSBORNE  AND  MENDEL:  The  growth  of  rats  upon  diets  of  isolated  food  sub- 
stances. Biochemical  Journal,  1916,  x,  534. 

OSBORNE,  T.  B.,  AND  MENDEL,  L.  B. :  Ophthalmia  and  diet.  J.  Am.  Med. 
Assn.,  1921,  Ixxvi,  905. 

OSBORNE,  T.  B.,  and  WAKEMAN,  A. :  Does  butter  fat  contain  N  and  P?  J. 
Biol.  Chem.,  1915,  xxi,  91. 

OSBORNE,  T.  B.,  AND  WAKEMAN,  A. :  The  proteins  of  cow's  milk.  J.  Biol. 
Chem.,  1918,  xxxiii,  7. 


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OSBORNE,  T.  B.,  AND  WAKEMAN,  A.:  Extraction  and  concentration  of  the 
water-soluble  vitamine  from  brewer's  yeast.  J.  Biol.  Chem., 
1919,  xl,  383. 

OSBORNE,  T.  B.,  AND  FERRY,  E. :  Preparation  of  protein  free  from  water- 
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PACINI  AND  RUSSELL:  The  presence  of  a  growth  producing  substance  in 
cultures  of  typhoid  bacilli.  J.  Biol.  Chem.,  1918,  xxxiv,  43. 

PADUA,  R.  G. :  Cystolithiasis  among  Filipinos  and  dietetic  deficiency.  Ph. 
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PALMER  AND  ECKLES:  Chemical  and  physiological  relation  of  pigment  of 
milk  fat  to  carotin  and  xanthophyll  of  green  plants.  J.  Biol. 
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PALMER  AND  ECKLES  :  The  pigments  of  the  body  fat,  corpus  luteum  and  the 
skin  secretions  of  the  cow.  J.  Biol.  Chem.,  1914,  xvii,  211. 

PALMER  AND  ECKLES:  The  yellow  lipochrome  of  the  blood  serum.  J.  Biol. 
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PALMER  AND  ECKLES  :  The  fate  of  carotin  and  xanthophyll  during  diges- 
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PALMER  AND  ECKLES:  The  pigments  of  human  milk  fat.  J.  Biol.  Chem., 
1914,  xvii,  245. 

PALMER  AND  COOLEDGE  :  Lactochrome.  The  yellow  pigment  of  milk  whey ; 
its  probable  identity  with  urochrome,  the  specific  yellow  pig- 
ment of  normal  urine.  J.  Biol.  Chem.,  1914,  xiv,  251. 

PALMER:  Xanthophyll,  the  principal  natural  yellow  pigment  of  the  egg 
yolk,  body  fat  and  blood  serum  of  the  hen.  J.  Biol.  Chem.,  1915, 
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PALMER:  The  physiological  relation  of  the  pigment  to  the  xanthophyll 
of  plants.  J.  Biol.  Chem.,  1915,  xvii,  261. 

PALMER  :  The  physiological  relation  of  plant  carotinoids  to  the  carotinoids 
of  the  cow,  horse,  sheep,  goat,  pig,  hen.  J.  Biol.  Chem.,  1916, 
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PALMER  AND  KEMPSTER:  The  relation  of  plant  carotinoids  to  growth, 
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AUG    2  1924 

SFP  m  1094 

.MAY  261925! 


OEC  20  1932 


IAK%  9  1938 

211938 

^291940 


1927 
928 
OCT  1  2  1929 


OCT  1  7 

MftR    22  1932 


2w-8,'21 


Library  of  the 
University  of  California  Medical  School  and  Hospitals 


